@article {pmid35623354, year = {2022}, author = {Johnson, MC and Hille, LT and Kleinstiver, BP and Meeske, AJ and Bondy-Denomy, J}, title = {Lack of Cas13a inhibition by anti-CRISPR proteins from Leptotrichia prophages.}, journal = {Molecular cell}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.molcel.2022.05.002}, pmid = {35623354}, issn = {1097-4164}, abstract = {CRISPR systems are prokaryotic adaptive immune systems that use RNA-guided Cas nucleases to recognize and destroy foreign genetic elements. To overcome CRISPR immunity, bacteriophages have evolved diverse families of anti-CRISPR proteins (Acrs). Recently, Lin et al. (2020) described the discovery and characterization of 7 Acr families (AcrVIA1-7) that inhibit type VI-A CRISPR systems. We detail several inconsistencies that question the results reported in the Lin et al. (2020) study. These include inaccurate bioinformatics analyses and bacterial strains that are impossible to construct. Published strains were provided by the authors, but MS2 bacteriophage plaque assays did not support the published results. We also independently tested the Acr sequences described in the original report, in E. coli and mammalian cells, but did not observe anti-Cas13a activity. Taken together, our data and analyses prompt us to question the claim that AcrVIA1-7 reported in Lin et al. are type VI anti-CRISPR proteins.}, }
@article {pmid35623245, year = {2022}, author = {Li, K and Luo, S and Guan, S and Situ, B and Wu, Y and Ou, Z and Tao, M and Zheng, L and Cai, Z}, title = {Tetrahedral framework nucleic acids linked CRISPR/Cas13a signal amplification system for rare tumor cell detection.}, journal = {Talanta}, volume = {247}, number = {}, pages = {123531}, doi = {10.1016/j.talanta.2022.123531}, pmid = {35623245}, issn = {1873-3573}, abstract = {The sensitive and accurate detection of rare tumor cells provides precise diagnosis and dynamic assessment information in various tumor spectrums. However, rare tumor cells assay is still a challenge due to the exceedingly rare presence in the blood. In this research, we develop a fluorescent approach for the identification of rare tumor cells based on a combination of immunosorbent capture and a three-step signal amplification strategy. First, rare tumor cells are captured by immunoadsorption on 96-well plates. Second, self-synthesized tetrahedral framework nucleic acids (tFNAs) spontaneously anchor into the lipid bilayer of rare tumor cells, resulting in a "one to more" amplification effect. Then, the double-stranded DNA (dsDNA) binds to the vertices of the tFNAs and generates a large amount of target RNA by T7 polymerase, which is the secondary signal amplification. Finally, the target RNA activates the collateral cleavage ability of CRISPR/Cas13a, and the reporter RNA is cleaved for third signal amplification. The detection limit of the proposed method is down to 1 cell mL-1. Furthermore, the tFNAs-Cas13a system is also shown to be capable of detecting rare tumor cells in spiked-in samples and clinical blood samples. This platform enables speedy detection of rare tumor cells with high sensitivity and good specificity, and shows great potential for tumor diagnosis.}, }
@article {pmid35620343, year = {2022}, author = {Akbari Kordkheyli, V and Rashidi, M and Shokri, Y and Fallahpour, S and Variji, A and Nabipour Ghara, E and Hosseini, SM}, title = {CRISPER/CAS System, a Novel Tool of Targeted Therapy of Drug-Resistant Lung Cancer.}, journal = {Advanced pharmaceutical bulletin}, volume = {12}, number = {2}, pages = {262-273}, doi = {10.34172/apb.2022.027}, pmid = {35620343}, issn = {2228-5881}, abstract = {Lung cancer (LC) is the most common cause of cancer-related death worldwide. Patients with LC are usually diagnosed at advanced phases. Five-year survival rate in LC patients is approximately 16%. Despite decades of research on LC treatments, clinical outcomes are still very poor, necessitating to develop novel technologies to manage the disease. Considering the role of genetic and epigenetic changes in oncogenes and tumor-suppressor genes in cancer progression, gene therapy provides a hot spot in cancer treatment research. Gene therapy offers less side effects compared to conventional methods such as chemotherapy. Unlike the traditional approaches of gene therapy that have temporary effects, using genetic modification tools can offer persistent cure. Over the past a few years, many studies have effectively used the CRISPR-Cas9 approach to modify gene expression in cells. This system is applied to induce site-specific mutagenesis and epigenetic modifications and regulate gene expression. In this review, we discuss recent applications of the CRISPR-Cas9 technology in treating LC.}, }
@article {pmid35613590, year = {2022}, author = {Del Amo, VL and Juste, SS and Gantz, VM}, title = {A nickase Cas9 gene-drive system promotes super-Mendelian inheritance in Drosophila.}, journal = {Cell reports}, volume = {39}, number = {8}, pages = {110843}, doi = {10.1016/j.celrep.2022.110843}, pmid = {35613590}, issn = {2211-1247}, mesh = {Animals ; CRISPR-Cas Systems/genetics ; DNA ; *Deoxyribonuclease I/metabolism ; Drosophila/metabolism ; *Gene Drive Technology ; Gene Editing ; }, abstract = {CRISPR-based gene-drives have been proposed for managing insect populations, including disease-transmitting mosquitoes, due to their ability to bias their inheritance toward super-Mendelian rates (>50%). Current technologies use a Cas9 that introduces DNA double-strand breaks into the opposing wild-type allele to replace it with a copy of the gene-drive allele via DNA homology-directed repair. However, the use of different Cas9 versions is unexplored, and alternative approaches could increase the available toolkit for gene-drive designs. Here, we report a gene-drive that relies on Cas9 nickases that generate staggered paired nicks in DNA to propagate the engineered gene-drive cassette. We show that generating 5' overhangs in the system yields efficient allelic conversion. The nickase gene-drive arrangement produces large, stereotyped deletions that are advantageous to eliminate viable animals carrying small mutations when targeting essential genes. Our nickase approach should expand the repertoire for gene-drive arrangements aimed at applications in mosquitoes and beyond.}, }
@article {pmid35618430, year = {2022}, author = {Woodside, WT and Vantsev, N and Catchpole, RJ and Garrett, SC and Olson, S and Graveley, BR and Terns, MP}, title = {Type III-A CRISPR-Cas systems as a versatile gene knockdown technology.}, journal = {RNA (New York, N.Y.)}, volume = {}, number = {}, pages = {}, doi = {10.1261/rna.079206.122}, pmid = {35618430}, issn = {1469-9001}, abstract = {CRISPR-Cas systems are functionally diverse prokaryotic anti-viral defense systems, which encompass six distinct types (I-VI) that each encode different effector Cas nucleases with distinct nucleic acid cleavage specificities. By harnessing the unique attributes of the various CRISPR-Cas systems, a range of innovative CRISPR-based DNA and RNA targeting tools and technologies have been developed. Here, we exploit the ability of type III-A CRISPR-Cas systems to carry out RNA-guided and sequence-specific target RNA cleavage for establishment of research tools for post-transcriptional control of gene expression. Type III-A systems from three bacterial species (L. lactis, S. epidermidis and S. thermophilus) were each expressed on a single plasmid in E. coli and the efficiency and specificity of gene knockdown was assessed by Northern blot and transcriptomic analysis. We show that engineered type III-A modules can be programmed using tailored CRISPR RNAs to efficiently knock down gene expression of both coding and non-coding RNAs in vivo. Moreover, simultaneous degradation of multiple cellular mRNA transcripts can be directed by utilizing a CRISPR array expressing corresponding gene-targeting crRNAs. Our results demonstrate the utility of distinct type III-A modules to serve as specific and effective gene knockdown platforms in heterologous cells. This transcriptome engineering technology has the potential to be further refined and exploited for key applications including gene discovery and gene pathway analyses in additional prokaryotic and perhaps eukaryotic cells and organisms.}, }
@article {pmid35617958, year = {2022}, author = {Omer-Javed, A and Pedrazzani, G and Albano, L and Ghaus, S and Latroche, C and Manzi, M and Ferrari, S and Fiumara, M and Jacob, A and Vavassori, V and Nonis, A and Canarutto, D and Naldini, L}, title = {Mobilization-based chemotherapy-free engraftment of gene-edited human hematopoietic stem cells.}, journal = {Cell}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.cell.2022.04.039}, pmid = {35617958}, issn = {1097-4172}, abstract = {Hematopoietic stem/progenitor cell gene therapy (HSPC-GT) is proving successful to treat several genetic diseases. HSPCs are mobilized, harvested, genetically corrected ex vivo, and infused, after the administration of toxic myeloablative conditioning to deplete the bone marrow (BM) for the modified cells. We show that mobilizers create an opportunity for seamless engraftment of exogenous cells, which effectively outcompete those mobilized, to repopulate the depleted BM. The competitive advantage results from the rescue during ex vivo culture of a detrimental impact of mobilization on HSPCs and can be further enhanced by the transient overexpression of engraftment effectors exploiting optimized mRNA-based delivery. We show the therapeutic efficacy in a mouse model of hyper IgM syndrome and further developed it in human hematochimeric mice, showing its applicability and versatility when coupled with gene transfer and editing strategies. Overall, our findings provide a potentially valuable strategy paving the way to broader and safer use of HSPC-GT.}, }
@article {pmid35616865, year = {2022}, author = {González, B and Vazquez-Vilar, M and Sánchez-Vicente, J and Orzáez, D}, title = {Optimization of Vectors and Targeting Strategies Including GoldenBraid and Genome Editing Tools: GoldenBraid Assembly of Multiplex CRISPR /Cas12a Guide RNAs for Gene Editing in Nicotiana benthamiana.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2480}, number = {}, pages = {193-214}, pmid = {35616865}, issn = {1940-6029}, abstract = {New breeding techniques, especially CRISPR/Cas, could facilitate the expansion and diversification of molecular farming crops by speeding up the introduction of new traits that improve their value as biofactories. One of the main advantages of CRISPR/Cas is its ability to target multiple loci simultaneously, a key feature known as multiplexing. This characteristic is especially relevant for polyploid species, as it is the case of Nicotiana benthamiana and other species of the same genus widely used in molecular farming. Here, we describe in detail the making of a multiplex DNA construct for genome editing in N. benthamiana using the GoldenBraid modular cloning platform. In this case, the procedure is adapted for the requirements of LbCas12a (Lachnospiraceae bacterium Cas12a), a nuclease whose cloning strategy differs from that of the more often used SpCas9 (Streptococcus pyogenes Cas9) enzyme. LbCas12a-mediated edition has several advantages, as its high editing efficiency, described for different plant species, and its T/A-rich PAM sequence, which expands the range of genomic loci that can be targeted by site-specific nucleases. The protocol also includes recommendations for the selection of protospacer sequences and indications for the analysis of editing results.}, }
@article {pmid35611733, year = {2022}, author = {Shang, L and Song, S and Zhang, T and Yan, K and Cai, H and Yuan, Y and Cheng, Y}, title = {[Propagation and phenotypic analysis of mutant rabbits with MSTN homozygous mutation].}, journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology}, volume = {38}, number = {5}, pages = {1847-1858}, doi = {10.13345/j.cjb.210541}, pmid = {35611733}, issn = {1872-2075}, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Muscle, Skeletal/metabolism ; Mutation ; *Myostatin/genetics/metabolism ; Phenotype ; Rabbits ; }, abstract = {Myostatin gene (MSTN) encodes a negative regulator for controlling skeletal muscle growth in animals. In this study, MSTN-/- homozygous mutants with "double muscle" phenotypic traits and stable inheritance were bred on the basis of MSTN gene editing rabbits, with the aim to establish a method for breeding homozygous progeny from primary MSTN biallelic mutant rabbits. MSTN-/- primary mutant rabbits were generated by CRISPR/Cas9 gene editing technology. The primary mutant rabbits were mated with wild type rabbits to produce F1 rabbits, whereas the F2 generation homozygous rabbits were bred by half-sibling mating or backcrossing with F1 generation rabbits of the same mutant strain. Sequence analysis of PCR products and its T vector cloning were used to screen homozygous rabbits. The MSTN mutant rabbits with 14-19 week-old were weighed and the difference of gluteus maximus tissue sections and muscle fiber cross-sectional area were calculated and analyzed. Five primary rabbits with MSTN gene mutation were obtained, among which three were used for homozygous breeding. A total of 15 homozygous rabbits (5 types of mutants) were obtained (M2-a: 3; M2-b: 2; M3-a: 2; M7-a: 6; M7-b: 2). The body weight of MSTN-/- homozygous mutant rabbits aged 14-19 weeks were significantly higher than that of MSTN+/+ wild-type rabbits of the same age ((2 718±120) g vs. (1 969±53) g, P < 0.01, a 38.0% increase). The mean cross sections of gluteus maximus muscle fiber in homozygous mutant rabbits were not only significantly higher than that of wild type rabbits ((3 512.2±439.2) μm2 vs. (1 274.8±327.3) μm2, P < 0.01), but also significantly higher than that of MSTN+/- hemizygous rabbits ((3 512.2±439.2) μm2 vs. (2 610.4±604.4) μm2, P < 0.05). In summary, five homozygous mutants rabbits of MSTN-/- gene were successfully bred, which showed a clear lean phenotype. The results showed that the primary breeds were non-chimeric mutant rabbits, and the mutant traits could be inherited from the offspring. MSTN-/- homozygous mutant rabbits of F2 generation could be obtained from F1 hemizygous rabbits by inbreeding or backcrossing. The progenies of the primary biallelic mutant rabbits were separated into two single-allelic mutants, both of which showed a "double-muscle" phenotype. Thus, this study has made progress in breeding high-quality livestock breeds with gene editing technology.}, }
@article {pmid35610381, year = {2022}, author = {Zhou, M and Cao, Y and Sui, M and Shu, X and Wan, F and Zhang, B}, title = {Dead Cas(t) light on new life: CRISPRa-mediated reprogramming of somatic cells into neurons.}, journal = {Cellular and molecular life sciences : CMLS}, volume = {79}, number = {6}, pages = {315}, pmid = {35610381}, issn = {1420-9071}, support = {81471283//National Natural Science Foundation of China/ ; 82072795//National Natural Science Foundation of China/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; *Gene Editing ; Neurons/metabolism ; Transcription Factors/genetics/metabolism ; }, abstract = {Overexpression of exogenous lineage-specific transcription factors could directly induce terminally differentiated somatic cells into target cell types. However, the low conversion efficiency and the concern about introducing exogenous genes limit the clinical application. With the rapid progress in genome editing, the application of CRISPR/dCas9 has been expanding rapidly, including converting somatic cells into other types of cells in vivo and in vitro. Using the CRISPR/dCas9 system, direct neuronal reprogramming could be achieved by activating endogenous genes. Here, we will discuss the latest progress, new insights, and future challenges of the application of the dCas9 system in direct neuronal reprogramming.}, }
@article {pmid35266285, year = {2022}, author = {Nayeri, S and Baghban Kohnehrouz, B and Ahmadikhah, A and Mahna, N}, title = {CRISPR/Cas9-mediated P-CR domain-specific engineering of CESA4 heterodimerization capacity alters cell wall architecture and improves saccharification efficiency in poplar.}, journal = {Plant biotechnology journal}, volume = {20}, number = {6}, pages = {1197-1212}, doi = {10.1111/pbi.13803}, pmid = {35266285}, issn = {1467-7652}, mesh = {*CRISPR-Cas Systems/genetics ; Cell Wall/genetics/metabolism ; Cellulose/metabolism ; Glucosyltransferases/genetics ; *Populus/genetics/metabolism ; }, abstract = {Cellulose is the most abundant unique biopolymer in nature with widespread applications in bioenergy and high-value bioproducts. The large transmembrane-localized cellulose synthase (CESA) complexes (CSCs) play a pivotal role in the biosynthesis and orientation of the para-crystalline cellulose microfibrils during secondary cell wall (SCW) deposition. However, the hub CESA subunit with high potential homo/heterodimerization capacity and its functional effects on cell wall architecture, cellulose crystallinity, and saccharification efficiency remains unclear. Here, we reported the highly potent binding site containing four residues of Pro435, Trp436, Pro437, and Gly438 in the plant-conserved region (P-CR) of PalCESA4 subunit, which are involved in the CESA4-CESA8 heterodimerization. The CRISPR/Cas9-knockout mutagenesis in the predicted binding site results in physiological abnormalities, stunt growth, and deficient roots. The homozygous double substitution of W436Q and P437S and heterozygous double deletions of W436 and P437 residues potentially reduced CESA4-binding affinity resulting in normal roots, 1.5-2-fold higher plant growth and cell wall regeneration rates, 1.7-fold thinner cell wall, high hemicellulose content, 37%-67% decrease in cellulose content, high cellulose DP, 25%-37% decrease in cellulose crystallinity, and 50% increase in saccharification efficiency. The heterozygous deletion of W436 increases about 2-fold CESA4 homo/heterodimerization capacity led to the 50% decrease in plant growth and increase in cell walls thickness, cellulose content (33%), cellulose DP (20%), and CrI (8%). Our findings provide a strategy for introducing commercial CRISPR/Cas9-mediated bioengineered poplars with promising cellulose applications. We anticipate our results could create an engineering revolution in bioenergy and cellulose-based nanomaterial technologies.}, }
@article {pmid35610585, year = {2022}, author = {Sun, L and Wang, J and Yan, F and Wang, G and Li, Y and Huang, J}, title = {CrisprVi: a software for visualizing and analyzing CRISPR sequences of prokaryotes.}, journal = {BMC bioinformatics}, volume = {23}, number = {Suppl 3}, pages = {172}, pmid = {35610585}, issn = {1471-2105}, support = {2018YFD0500500//National Key Research and Development Program of China/ ; R1805//Open Project Program of Jiangsu Key Laboratory of Zoonosis/ ; BK20190878//Natural Science Foundation of Jiangsu Province/ ; yzuxk202015//Cross-Disciplinary Project of the Animal Science Special Discipline of Yangzhou University/ ; }, abstract = {BACKGROUND: Clustered regularly interspaced short palindromic repeats (CRISPR) and their spacers are important components of prokaryotic CRISPR-Cas systems. In order to analyze the CRISPR loci of multiple genomes more intuitively and comparatively, here we propose a visualization analysis tool named CrisprVi.<br><br>RESULTS: CrisprVi is a Python package consisting of a graphic user interface (GUI) for visualization, a module for commands parsing and data transmission, local SQLite and BLAST databases for data storage and a functions layer for data processing. CrisprVi can not only visually present information of CRISPR direct repeats (DRs) and spacers, such as their orders on the genome, IDs, start and end coordinates, but also provide interactive operation for users to display, label and align the CRISPR sequences, which help researchers investigate the locations, orders and components of the CRISPR sequences in a global view. In comparison to other CRISPR visualization tools such as CRISPRviz and CRISPRStudio, CrisprVi not only improves the interactivity and effects of the visualization, but also provides basic statistics of the CRISPR sequences, and the consensus sequences of DRs/spacers across the input strains can be inspected from a clustering heatmap based on the BLAST results of the CRISPR sequences hitting against the genomes.<br><br>CONCLUSIONS: CrisprVi is a convenient tool for visualizing and analyzing the CRISPR sequences and it would be helpful for users to inspect novel CRISPR-Cas systems of prokaryotes.}, }
@article {pmid35609453, year = {2022}, author = {Wu, H and Cao, X and Meng, Y and Richards, D and Wu, J and Ye, Z and deMello, AJ}, title = {DropCRISPR: A LAMP-Cas12a based digital method for ultrasensitive detection of nucleic acid.}, journal = {Biosensors &amp; bioelectronics}, volume = {211}, number = {}, pages = {114377}, doi = {10.1016/j.bios.2022.114377}, pmid = {35609453}, issn = {1873-4235}, abstract = {Since their discovery, CRISPR/Cas systems have been extensively exploited in nucleic acid biosensing. However, the vast majority of contemporary platforms offer only qualitative detection of nucleic acid, and fail to realize ultrasensitive quantitative detection. Herein, we report a digital droplet-based platform (DropCRISPR), which combines loop-mediated isothermal amplification (LAMP) with CRISPR/Cas12a to realize ultrasensitive and quantitative detection of nucleic acids. This is achieved through a novel two-step microfluidic system which combines droplet LAMP with a picoinjector capable of injecting the required CRISPR/Cas12a reagents into each droplet. This method circumvents the temperature incompatibilities of LAMP and CRISPR/Cas12a and avoids mutual interference between amplification reaction and CRISPR detection. Ultrasensitive detection (at fM level) was achieved for a model plasmid containing the invA gene of Salmonella typhimurium (St), with detection down to 102 cfu/mL being achieved in pure bacterial culture. Additionally, we demonstrate that the DropCRISPR platform is capable of detecting St in raw milk samples without additional nucleic acid extraction. The sensitivity and robustness of the DropCRISPR further demonstrates the potential of CRISPR/Cas-based diagnostic platforms, particularly when combined with state-of-the-art microfluidic architectures.}, }
@article {pmid35608343, year = {2022}, author = {Zhang, C and Li, N and Rao, L and Li, J and Liu, Q and Tian, C}, title = {Development of an Efficient C-to-T Base-Editing System and Its Application to Cellulase Transcription Factor Precise Engineering in Thermophilic Fungus Myceliophthora thermophila.}, journal = {Microbiology spectrum}, volume = {}, number = {}, pages = {e0232121}, doi = {10.1128/spectrum.02321-21}, pmid = {35608343}, issn = {2165-0497}, abstract = {Myceliophthora thermophila is a thermophilic fungus with great potential in biorefineries and biotechnology. The base editor is an upgraded version of the clustered regularly interspaced short palindromic repeats (CRISPR)-dependent genome-editing tool that introduces precise point mutations without causing DNA double-strand breaks (DSBs) and has been used in various organisms but rarely in filamentous fungi, especially thermophilic filamentous fungi. Here, for the first time, we constructed three cytosine base editors (CBEs) in M. thermophila, namely, evolved apolipoprotein B mRNA-editing enzyme catalytic subunit 1 (APOBEC1) cytosine base editor 4 max (Mtevo-BE4max), bacteriophage Mu Gam protein cytosine base editor 4 max (MtGAM-BE4max), and evolved CDA1 deaminase cytosine base editor (Mtevo-CDA1), and efficiently inactivated genes by precisely converting three codons (CAA, CAG, and CGA) into stop codons without DSB formation. The Mtevo-CDA1 editor with up to 92.6% editing efficiency is a more suitable tool for cytosine base editing in thermophilic fungi. To investigate the function of each motif of the cellulase transcription factor M. thermophila CLR-2 (MtCLR-2), we used the Mtevo-CDA1 editor. The fungal-specific motif of MtCLR-2 was found to be strongly involved in cellulase secretion, conidium formation, hyphal branching, and colony formation. Mutation of the fungus-specific motif caused significant defects in these characteristics. Thus, we developed an efficient thermophilic fungus-compatible base-editing system that could also be used for genetic engineering in other relevant filamentous fungi. IMPORTANCE A CRISPR/Cas-based base-editing approach has been developed to introduce point mutations without inducing double-strand breaks (DSBs) and attracted substantial academic and industrial interest. Our study developed the deaminase-cytosine base-editing system to efficiently edit three target genes, amdS, cre-1, and the essential cellulase regulator gene Mtclr-2, in Myceliophthora thermophila. A variety of point mutations in the target loci of the DNA-binding domain and fungus-specific motif of M. thermophila CLR-2 (MtCLR-2) were successfully generated via our base editor Mtevo-CDA1 to elucidate its function. Here, we show that the DNA-binding domain of MtCLR-2 is important for the fungal response to cellulose conditions, while its fungus-specific motif is involved in fungal growth. These findings indicate that our base editor can be an effective tool for elucidating the functions of motifs of target genes in filamentous fungi and for metabolic engineering in the field of synthetic biology.}, }
@article {pmid35606745, year = {2022}, author = {Metzloff, M and Yang, E and Dhole, S and Clark, AG and Messer, PW and Champer, J}, title = {Experimental demonstration of tethered gene drive systems for confined population modification or suppression.}, journal = {BMC biology}, volume = {20}, number = {1}, pages = {119}, pmid = {35606745}, issn = {1741-7007}, support = {R21AI130635/NH/NIH HHS/United States ; R01GM127418/NH/NIH HHS/United States ; F32AI138476/NH/NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Drosophila/genetics ; *Gene Drive Technology/methods ; }, abstract = {BACKGROUND: Homing gene drives hold great promise for the genetic control of natural populations. However, current homing systems are capable of spreading uncontrollably between populations connected by even marginal levels of migration. This could represent a substantial sociopolitical barrier to the testing or deployment of such drives and may generally be undesirable when the objective is only local population control, such as suppression of an invasive species outside of its native range. Tethered drive systems, in which a locally confined gene drive provides the CRISPR nuclease needed for a homing drive, could provide a solution to this problem, offering the power of a homing drive and confinement of the supporting drive.<br><br>RESULTS: Here, we demonstrate the engineering of a tethered drive system in Drosophila, using a regionally confined CRISPR Toxin-Antidote Recessive Embryo (TARE) drive to support modification and suppression homing drives. Each drive was able to bias inheritance in its favor, and the TARE drive was shown to spread only when released above a threshold frequency in experimental cage populations. After the TARE drive had established in the population, it facilitated the spread of a subsequently released split homing modification drive (to all individuals in the cage) and of a homing suppression drive (to its equilibrium frequency).<br><br>CONCLUSIONS: Our results show that the tethered drive strategy is a viable and easily engineered option for providing confinement of homing drives to target populations.}, }
@article {pmid35596077, year = {2022}, author = {Pan, C and Li, G and Malzahn, AA and Cheng, Y and Leyson, B and Sretenovic, S and Gurel, F and Coleman, GD and Qi, Y}, title = {Boosting plant genome editing with a versatile CRISPR-Combo system.}, journal = {Nature plants}, volume = {8}, number = {5}, pages = {513-525}, pmid = {35596077}, issn = {2055-0278}, support = {IOS-1758745//National Science Foundation (NSF)/ ; }, mesh = {*Arabidopsis/genetics ; CRISPR-Cas Systems ; *Gene Editing ; Genome, Plant ; Plant Breeding ; Plants, Genetically Modified/genetics ; }, abstract = {CRISPR-Cas9, its derived base editors and CRISPR activation systems have greatly aided genome engineering in plants. However, these systems are mostly used separately, leaving their combinational potential largely untapped. Here we develop a versatile CRISPR-Combo platform, based on a single Cas9 protein, for simultaneous genome editing (targeted mutagenesis or base editing) and gene activation in plants. We showcase the powerful applications of CRISPR-Combo for boosting plant genome editing. First, CRISPR-Combo is used to shorten the plant life cycle and reduce the efforts in screening transgene-free genome-edited plants by activation of a florigen gene in Arabidopsis. Next, we demonstrate accelerated regeneration and propagation of genome-edited plants by activation of morphogenic genes in poplar. Furthermore, we apply CRISPR-Combo to achieve rice regeneration without exogenous plant hormones, which is established as a new method to predominately enrich heritable targeted mutations. In conclusion, CRISPR-Combo is a versatile genome engineering tool with promising applications in crop breeding.}, }
@article {pmid35595842, year = {2022}, author = {Flegler, A and Lipski, A}, title = {Engineered CRISPR/Cas9 System for Transcriptional Gene Silencing in Arthrobacter Species Indicates Bacterioruberin is Indispensable for Growth at Low Temperatures.}, journal = {Current microbiology}, volume = {79}, number = {7}, pages = {199}, pmid = {35595842}, issn = {1432-0991}, mesh = {*Arthrobacter/genetics/metabolism ; CRISPR-Cas Systems ; Carotenoids/metabolism ; Temperature ; }, abstract = {Pink-pigmented Arthrobacter species produce the rare C50 carotenoid bacterioruberin, which is suspected to be part of the cold adaptation mechanism. In silico analysis of the repertoire of genes encoded by the Arthrobacter agilis and Arthrobacter bussei genome revealed the biosynthetic pathway of bacterioruberin. Although genetic analysis is an essential tool for studying the physiology of Arthrobacter species, genetic manipulation of Arthrobacter is always time and labor intensive due to the lack of genetic engineering tools. Here we report the construction and application of a CRISPR/deadCas9 system (pCasiART) for gene silencing in Arthrobacter species. The engineered system pCasiART is suitable for the Golden Gate assembly of spacers, enabling rapid and accurate construction of adapted systems. In addition, pCasiART has been developed to provide an efficient transcription inhibition system for genome-wide gene silencing. The gene silencing of the phytoene synthase (CrtB), the first enzyme in bacterioruberin biosynthesis, suppressed bacterioruberin biosynthesis in Arthrobacter agilis and Arthrobacter bussei, resulting in a lack of pink pigmentation, reduction of biomass production, and growth rates at low temperatures.}, }
@article {pmid35549163, year = {2022}, author = {Zeng, R and Gong, H and Li, Y and Li, Y and Lin, W and Tang, D and Knopp, D}, title = {CRISPR-Cas12a-Derived Photoelectrochemical Biosensor for Point-Of-Care Diagnosis of Nucleic Acid.}, journal = {Analytical chemistry}, volume = {94}, number = {20}, pages = {7442-7448}, doi = {10.1021/acs.analchem.2c01373}, pmid = {35549163}, issn = {1520-6882}, mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems ; Electrochemical Techniques ; Humans ; Hydrogen Peroxide ; *Nucleic Acids ; Point-of-Care Systems ; }, abstract = {This work presented a point-of-care (POC) photoelectrochemical (PEC) biosensing for the detection of human papillomavirus-16 (HPV-16) on a portable electrochemical detection system by using CRISPR-Cas12a trans-cleaving the G-quadruplex for the biorecognition/amplification and a hollow In2O3-In2S3-modified screen-printed electrode (In2O3-In2S3/SPE) as the photoactive material. G-quadruplexes were capable of biocatalytic precipitation (H2O2-mediated 4-chloro-1-naphthol oxidation) on the In2O3-In2S3/SPE surface, resulting in a weakened photocurrent, but suffered from trans-cleavage when the CRISPR-Cas12a system specifically recognized the analyte. The photocurrent results could be directly observed with the card-sized electrochemical device via a smartphone, which displayed a high-value photocurrent for these positive samples, while a low-value photocurrent for the target-free samples. Such a system exhibited satisfying photocurrent responses toward HPV-16 within a wide working range from 5.0 to 5000 pM and allowed for detection of HPV-16 at a concentration as low as 1.2 pM. The proposed assay provided a smartphone signal readout to enable the rapid screening PEC determination of HPV-16 concentration without sophisticated instruments, thus meeting the requirements of remote areas and resource-limited settings. We envision that combining an efficient biometric PEC sensing platform with a wireless card-sized electrochemical device will enable high-throughput POC diagnostic analysis.}, }
@article {pmid35544322, year = {2022}, author = {Liang, Y and Xie, J and Zhang, Q and Wang, X and Gou, S and Lin, L and Chen, T and Ge, W and Zhuang, Z and Lian, M and Chen, F and Li, N and Ouyang, Z and Lai, C and Liu, X and Li, L and Ye, Y and Wu, H and Wang, K and Lai, L}, title = {AGBE: a dual deaminase-mediated base editor by fusing CGBE with ABE for creating a saturated mutant population with multiple editing patterns.}, journal = {Nucleic acids research}, volume = {50}, number = {9}, pages = {5384-5399}, pmid = {35544322}, issn = {1362-4962}, support = {81941004//National Natural Science Foundation of China/ ; 2021YFA0805903//National Key Research and Development Program of China/ ; ZDYF2021SHFZ052//Key Research &amp; Development Program of Hainan Province/ ; ZDKJ2021030//Major Science and Technology Project of Hainan Province/ ; KFJ-BRP-017-57//Chinese Academy of Sciences/ ; 2019347//Youth Innovation Promotion Association of the Chinese Academy of Sciences/ ; 2018GZR110104004//Key Research &amp; Development Program of Bioland Laboratory/ ; 2020B1212060052//Science and Technology Planning Project of Guangdong Province/ ; 202007030003//Science and Technology Program of Guangzhou, China/ ; 202002011//2020 Research Program of Sanya Yazhou Bay Science and Technology City/ ; 2019-I2M-5-025//Chinese Academy of Medical Sciences/ ; YESS20200024//CAST/ ; }, mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; INDEL Mutation ; Mammals/genetics ; Mutation ; Uracil-DNA Glycosidase/genetics ; }, abstract = {Establishing saturated mutagenesis in a specific gene through gene editing is an efficient approach for identifying the relationships between mutations and the corresponding phenotypes. CRISPR/Cas9-based sgRNA library screening often creates indel mutations with multiple nucleotides. Single base editors and dual deaminase-mediated base editors can achieve only one and two types of base substitutions, respectively. A new glycosylase base editor (CGBE) system, in which the uracil glycosylase inhibitor (UGI) is replaced with uracil-DNA glycosylase (UNG), was recently reported to efficiently induce multiple base conversions, including C-to-G, C-to-T and C-to-A. In this study, we fused a CGBE with ABE to develop a new type of dual deaminase-mediated base editing system, the AGBE system, that can simultaneously introduce 4 types of base conversions (C-to-G, C-to-T, C-to-A and A-to-G) as well as indels with a single sgRNA in mammalian cells. AGBEs can be used to establish saturated mutant populations for verification of the functions and consequences of multiple gene mutation patterns, including single-nucleotide variants (SNVs) and indels, through high-throughput screening.}, }
@article {pmid35483302, year = {2022}, author = {Li, Y and Yang, F and Yuan, R and Zhong, X and Zhuo, Y}, title = {Electrochemiluminescence covalent organic framework coupling with CRISPR/Cas12a-mediated biosensor for pesticide residue detection.}, journal = {Food chemistry}, volume = {389}, number = {}, pages = {133049}, doi = {10.1016/j.foodchem.2022.133049}, pmid = {35483302}, issn = {1873-7072}, mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems ; DNA/genetics ; *Metal-Organic Frameworks ; *Pesticide Residues ; }, abstract = {The trace detection of pesticide residue becomes particularly important since increasing attentions have been attached to food safety. Herein, we developed an electrochemiluminescence (ECL) covalent organic framework (COF) based-biosensor for trace pesticide detection coupling with CRISPR/Cas12a-mediated signal accumulation strategy. Firstly, the target conversion was carried out with an aptamer-assembled magnetic spherical nucleic acids, which can convert acetamiprid to activator DNA, triggering the CRISPR/Cas12a to make quenching probes far away from electrode for signal accumulation. The COF with stable and strong ECL was synthesized by a condensation reaction between the perylene-3,4,9,10-tetracarboxylic dianhydride (PTCDA) and melamine (MA), due to the highly ordered arrangement of the PTCDA luminescence units among COF structure and the pore confinement effect. Moreover, the designed assay method was successfully employed to detect the residual level of acetamiprid in real sample and expected to be widely used in pesticide-related food safety.}, }
@article {pmid35418245, year = {2022}, author = {Parashar, A and Bak, K and Murshed, M}, title = {Prevention of Arterial Elastocalcinosis: Differential Roles of the Conserved Glutamic Acid and Serine Residues of Matrix Gla Protein.}, journal = {Arteriosclerosis, thrombosis, and vascular biology}, volume = {42}, number = {6}, pages = {e155-e167}, doi = {10.1161/ATVBAHA.122.317518}, pmid = {35418245}, issn = {1524-4636}, abstract = {BACKGROUND: Inactivating mutations in matrix Gla protein (MGP) lead to Keutel syndrome, a rare disease hallmarked by ectopic calcification of cartilage and vascular tissues. Although MGP acts as a strong inhibitor of arterial elastic lamina calcification (elastocalcinosis), its mode of action is unknown. Two sets of conserved residues undergoing posttranslational modifications-4 glutamic acid residues, which are γ-carboxylated by gamma-glutamyl carboxylase; and 3 serine residues, which are phosphorylated by yet unknown kinase(s)-are thought to be essential for MGP's function.<br><br>METHODS: We pursued a genetic approach to study the roles of MGP's conserved residues. First, a transgenic line (SM22a-GlamutMgp) expressing a mutant form of MGP, in which the conserved glutamic acid residues were mutated to alanine, was generated. The transgene was introduced to Mgp-/- mice to generate a compound mutant, which produced the mutated MGP only in the vascular tissues. We generated a second mouse model (MgpS3mut/S3mut) to mutate MGP's conserved serine residues to alanine. The initiation and progression of vascular calcification in these models were analyzed by alizarin red staining, histology, and micro-computed tomography imaging.<br><br>RESULTS: On a regular diet, the arterial walls in the Mgp-/-; SM22α-GlamutMgp mice were not calcified. However, on a high phosphorus diet, these mice showed wide-spread arterial calcification. In contrast, MgpS3mut/S3mut mice on a regular diet recapitulated arterial calcification traits of Mgp-/- mice, although with lesser severity.<br><br>CONCLUSIONS: For the first time, we show here that MGP's conserved serine residues are indispensable for its antimineralization function in the arterial tissues. Although the conserved glutamic acid residues are not essential for this function on a regular diet, they are needed to prevent phosphate-induced arterial elastocalcinosis.}, }
@article {pmid35378143, year = {2022}, author = {Kovářová, J and Novotná, M and Faria, J and Rico, E and Wallace, C and Zoltner, M and Field, MC and Horn, D}, title = {CRISPR/Cas9-based precision tagging of essential genes in bloodstream form African trypanosomes.}, journal = {Molecular and biochemical parasitology}, volume = {249}, number = {}, pages = {111476}, doi = {10.1016/j.molbiopara.2022.111476}, pmid = {35378143}, issn = {1872-9428}, mesh = {CRISPR-Cas Systems ; Genes, Essential ; *Trypanosoma/genetics ; *Trypanosoma brucei brucei/genetics ; Untranslated Regions ; }, abstract = {Proteins of interest are frequently expressed with a fusion-tag to facilitate experimental analysis. In trypanosomatids, which are typically diploid, a tag-encoding DNA fragment is typically fused to one native allele. However, since recombinant cells represent ≪0.1% of the population following transfection, these DNA fragments also incorporate a marker cassette for positive selection. Consequently, native mRNA untranslated regions (UTRs) are replaced, potentially perturbing gene expression; in trypanosomatids, UTRs often impact gene expression in the context of widespread and constitutive polycistronic transcription. We sought to develop a tagging strategy that preserves native UTRs in bloodstream-form African trypanosomes, and here we describe a CRISPR/Cas9-based knock-in approach to drive precise and marker-free tagging of essential genes. Using simple tag-encoding amplicons, we tagged four proteins: a histone acetyltransferase, HAT2; a histone deacetylase, HDAC3; a cleavage and polyadenylation specificity factor, CPSF3; and a variant surface glycoprotein exclusion factor, VEX2. The approach maintained the native UTRs and yielded clonal strains expressing functional recombinant proteins, typically with both alleles tagged. We demonstrate utility for both immunofluorescence-based localisation and for enriching protein complexes; GFPHAT2 or GFPHDAC3 complexes in this case. This precision tagging approach facilitates the assembly of strains expressing essential recombinant genes with their native UTRs preserved.}, }
@article {pmid35304271, year = {2022}, author = {Wen, W and Zhang, XB}, title = {CRISPR-Cas9 gene editing induced complex on-target outcomes in human cells.}, journal = {Experimental hematology}, volume = {110}, number = {}, pages = {13-19}, doi = {10.1016/j.exphem.2022.03.002}, pmid = {35304271}, issn = {1873-2399}, mesh = {*CRISPR-Cas Systems ; DNA Repair ; *Gene Editing/methods ; Genetic Therapy ; Genome ; Humans ; }, abstract = {CRISPR-Cas9 is a powerful tool for editing the genome and holds great promise for gene therapy applications. Initial concerns of gene engineering focus on off-target effects. However, in addition to short indel mutations (often <50 bp), an increasing number of studies have revealed complex on-target results after double-strand break repair by CRISPR-Cas9, such as large deletions, gene rearrangement, and loss of heterozygosity. These unintended mutations are potential safety concerns in clinical gene editing. Here, in this review, we summarize the significant findings of CRISPR-Cas9-induced on-target deleterious outcomes and discuss putative ways to achieve safe gene therapy.}, }
@article {pmid35247491, year = {2022}, author = {Chae, SY and Jeong, E and Kang, S and Yim, Y and Kim, JS and Min, DH}, title = {Rationally designed nanoparticle delivery of Cas9 ribonucleoprotein for effective gene editing.}, journal = {Journal of controlled release : official journal of the Controlled Release Society}, volume = {345}, number = {}, pages = {108-119}, doi = {10.1016/j.jconrel.2022.02.035}, pmid = {35247491}, issn = {1873-4995}, mesh = {CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; *Gene Editing/methods ; *Nanoparticles ; Ribonucleoproteins/genetics ; }, abstract = {Programmable endonucleases such as CRISPR/Cas9 system emerge as a promising tool to treat genetic and non-genetic diseases such as hypercholesterolemia, Duchenne muscular dystrophy, and cancer. However, the lack of safe and efficient vehicles that enable intracellular delivery of CRISPR/Cas9 endonuclease is a big hurdle for its therapeutic applications. Here, we employed porous nanoparticle for the Cas9 ribonucleoprotein (RNP) delivery and achieved efficient knockout of target genes in vitro and in vivo. The porous nanoparticle, called 'BALL', enabled safe and direct intracellular Cas9 RNP delivery by improving bioavailability and serum stability. The BALL-mediated delivery of Cas9 RNP showed superior indel efficiency of about 40% in vitro and 20% in vivo in a model system employing green fluorescent protein (GFP). More importantly, intramuscular injection of the Cas9 RNP-BALL complex targeting the myostatin (MSTN) gene which is known to suppress muscle growth achieved successful knockout of the MSTN gene, resulting in the increase of muscle and the improved motor functions. Thus, we believe that the BALL is a promising delivery system for CRISPR-based genome editing technology, which can be applied to the treatment of various genetic diseases.}, }
@article {pmid34951457, year = {2022}, author = {Paul, B and Chaubet, L and Verver, DE and Montoya, G}, title = {Mechanics of CRISPR-Cas12a and engineered variants on λ-DNA.}, journal = {Nucleic acids research}, volume = {50}, number = {9}, pages = {5208-5225}, pmid = {34951457}, issn = {1362-4962}, support = {NNF14CC0001//Novo Nordisk Foundation/ ; NNF18OC0055061//Distinguished Investigator/ ; }, mesh = {*CRISPR-Associated Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; DNA/chemistry ; DNA, Single-Stranded/genetics ; Endonucleases/metabolism ; Gene Editing ; RNA, Guide/genetics ; }, abstract = {Cas12a is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here, we selected a target site on bacteriophage λ-DNA and used optical tweezers combined with fluorescence to provide mechanistic insight into wild type Cas12a and three engineered variants, where the specific dsDNA and the unspecific ssDNA cleavage are dissociated (M1 and M2) and a third one which nicks the target DNA (M3). At low forces wtCas12a and the variants display two main off-target binding sites, while on stretched dsDNA at higher forces numerous binding events appear driven by the mechanical distortion of the DNA and partial matches to the crRNA. The multiple binding events onto dsDNA at high tension do not lead to cleavage, which is observed on the target site at low forces when the DNA is flexible. In addition, activity assays also show that the preferential off-target sites for this crRNA are not cleaved by wtCas12a, indicating that λ-DNA is only severed at the target site. Our single molecule data indicate that the Cas12a scaffold presents singular mechanical properties, which could be used to generate new endonucleases with biomedical and biotechnological applications.}, }
@article {pmid35605545, year = {2022}, author = {Jia, HY and Zhao, HL and Wang, T and Chen, PR and Yin, BC and Ye, BC}, title = {A programmable and sensitive CRISPR/Cas12a-based MicroRNA detection platform combined with hybridization chain reaction.}, journal = {Biosensors &amp; bioelectronics}, volume = {211}, number = {}, pages = {114382}, doi = {10.1016/j.bios.2022.114382}, pmid = {35605545}, issn = {1873-4235}, abstract = {MicroRNAs (miRNAs) play an essential role in cancer diagnosis and prognosis. Developing a new method for sensitive detection of miRNA is constantly in demand. CRISPR/Cas12a system can nonspecifically cleave single-stranded DNA after specific recognition of target DNA, showing tremendous potential in molecular diagnostics. However, CRISPR-based detection methods require synthesizing different crRNAs for detecting different targets, which limit their widespread application. Herein, we design a versatile and sensitive miRNA detection platform based on CRISPR/Cas12a system combined with a hybridization chain reaction (HCR) circuit. In this design, the HCR circuit as the signal transducer converts each miRNA into multiple DNA duplexes, which act as the activators to activate the trans-cleavage activity of Cas12a for further signal amplification. More importantly, this platform can sensitively detect different miRNAs without changing the spacer sequence of crRNA due to the fixed activators formed by HCR. In addition, the consistency between the proposed platform and RT-qPCR in miRNA detection extracted from different cell lines validated its practicability, demonstrating the potential in clinical diagnosis of cancers and monitoring therapy.}, }
@article {pmid35589728, year = {2022}, author = {Yuan, Q and Gao, X}, title = {Multiplex base- and prime-editing with drive-and-process CRISPR arrays.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {2771}, pmid = {35589728}, issn = {2041-1723}, support = {R01HL157714//U.S. Department of Health &amp; Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; }, mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; Humans ; RNA, Guide/genetics ; RNA, Transfer/genetics ; }, abstract = {Current base- and prime-editing technologies lack efficient strategies to edit multiple genomic loci simultaneously, limiting their applications in complex genomics and polygenic diseases. Here, we describe drive-and-process (DAP) CRISPR array architectures for multiplex base-editing (MBE) and multiplex prime-editing (MPE) in human cells. We leverage tRNA as the RNA polymerase III promoter to drive the expression of tandemly assembled tRNA-guide RNA (gRNA) arrays, of which the individual gRNAs are released by the cellular endogenous tRNA processing machinery. We engineer a 75-nt human cysteine tRNA (hCtRNA) for the DAP array, achieving up to 31-loci MBE and up to 3-loci MPE. By applying MBE or MPE elements for deliveries via adeno-associated virus (AAV) and lentivirus, we demonstrate simultaneous editing of multiple disease-relevant genomic loci. Our work streamlines the expression and processing of gRNAs on a single array and establishes efficient MBE and MPE strategies for biomedical research and therapeutic applications.}, }
@article {pmid35545708, year = {2022}, author = {Tang, L}, title = {Spatial CRISPR screens in tumors.}, journal = {Nature methods}, volume = {19}, number = {5}, pages = {517}, pmid = {35545708}, issn = {1548-7105}, mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Humans ; *Neoplasms/genetics ; RNA, Guide ; }, }
@article {pmid35481381, year = {2022}, author = {Liang, Y and Zou, L and Lin, H and Li, B and Zhao, J and Wang, H and Sun, J and Chen, J and Mo, Y and Yang, X and Deng, X and Tang, S}, title = {Detection of Major SARS-CoV-2 Variants of Concern in Clinical Samples via CRISPR-Cas12a-Mediated Mutation-Specific Assay.}, journal = {ACS synthetic biology}, volume = {11}, number = {5}, pages = {1811-1823}, doi = {10.1021/acssynbio.1c00643}, pmid = {35481381}, issn = {2161-5063}, mesh = {*COVID-19/diagnosis ; CRISPR-Cas Systems/genetics ; Humans ; Mutation ; *SARS-CoV-2/genetics ; }, abstract = {Objectives: Emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants pose a great threat and burden to global public health. Here, we evaluated a clustered regularly interspaced short palindromic repeat-associated enzyme 12a (CRISPR-Cas12a)-based method for detecting major SARS-CoV-2 variants of concern (VOCs) in SARS-CoV-2 positive clinical samples. Methods: Allele-specific CRISPR RNAs (crRNAs) targeting the signature mutations in the spike protein of SARS-CoV-2 are designed. A total of 59 SARS-CoV-2 positive oropharyngeal swab specimens were used to evaluate the performance of the CRISPR-Cas12a-mediated assay to identify major SARS-CoV-2 VOCs. Results: Compared with Sanger sequencing, the eight allele-specific crRNAs analyzed can specifically identify the corresponding mutations with a positive predictive value of 83.3-100% and a negative predictive value of 85.7-100%. Our CRISPR-Cas12a-mediated assay distinguished wild-type and four major VOCs (Alpha, Beta, Delta, and Omicron) of SARS-CoV-2 with a sensitivity of 93.8-100.0% and a specificity of 100.0%. The two methods showed a concordance of 98.3% (58/59) with a κ value of 0.956-1.000, while seven (11.9%) samples were found to be positive for extra mutations by the CRISPR-based assay. Furthermore, neither virus titers nor the sequences adjacent to the signature mutations were associated with the variation of fluorescence intensity detected or the false-positive reaction observed when testing clinical samples. In addition, there was no cross-reaction observed when detecting 33 SARS-CoV-2 negative clinical samples infected with common respiratory pathogens. Conclusions: The CRISPR-Cas12a-based genotyping assay is highly sensitive and specific when detecting both the SARS-CoV-2 wild-type strain and major VOCs. It is a simple and rapid assay that can monitor and track the circulating SARS-CoV-2 variants and the dynamics of the coronavirus disease 2019 (COVID-19) pandemic and can be easily implemented in resource-limited settings.}, }
@article {pmid35471824, year = {2022}, author = {Lei, R and Li, L and Wu, P and Fei, X and Zhang, Y and Wang, J and Zhang, D and Zhang, Q and Yang, N and Wang, X}, title = {RPA/CRISPR/Cas12a-Based On-Site and Rapid Nucleic Acid Detection of Toxoplasma gondii in the Environment.}, journal = {ACS synthetic biology}, volume = {11}, number = {5}, pages = {1772-1781}, doi = {10.1021/acssynbio.1c00620}, pmid = {35471824}, issn = {2161-5063}, mesh = {CRISPR-Cas Systems/genetics ; Humans ; Nucleic Acid Amplification Techniques ; *Nucleic Acids ; Recombinases/genetics/metabolism ; Sensitivity and Specificity ; *Toxoplasma/genetics/metabolism ; }, abstract = {Toxoplasma gondii is an opportunistic pathogen widely distributed within the world, poses a huge threat to human health, and causes significant economic losses to the livestock industry. Herein, we developed a portable one-pot detection of T. gondii by combining recombinase polymerase amplification (RPA) and a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system. A glass microfiber filter device used for the first step can efficiently extract T. gondii from low-concentration samples. The lyophilized RPA reagents and Cas12a/crRNA reagents are prestored in one Eppendorf tube, and both reactions can be performed on a low-cost thermal controller (∼37 °C), avoiding the drawbacks of the step-by-step addition of components. The developed RPA/CRISPR/Cas12a system exhibits a high selectivity toward the B1 gene amplicon of T. gondii over other parasites with a limit of detection of 3.3 copies/μL. The visual signal readout can be easily realized by a fluorometer or lateral-flow strip. A portable suitcase containing the minimum equipment and lyophilized reagents was adopted for the rapid determination of T. gondii in heavily polluted landfill leachate. This system presents rapidness, robustness and on-site features for the detection of nucleic acids of the parasite, making it a promising tool for field applications in remote areas.}, }
@article {pmid35471009, year = {2022}, author = {Zhu, X and Wu, Y and Lv, X and Liu, Y and Du, G and Li, J and Liu, L}, title = {Combining CRISPR-Cpf1 and Recombineering Facilitates Fast and Efficient Genome Editing in Escherichia coli.}, journal = {ACS synthetic biology}, volume = {11}, number = {5}, pages = {1897-1907}, doi = {10.1021/acssynbio.2c00041}, pmid = {35471009}, issn = {2161-5063}, mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Escherichia coli/genetics ; *Gene Editing/methods ; Histidine/genetics ; Metabolic Engineering/methods ; }, abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)-based gene-editing technology has been widely used in various microorganisms due to its advantages of low cost, high efficiency, easy operation, and multiple functions. In this study, an efficient and fast double-plasmid gene-editing system pEcCpf1/pcrEG was constructed in Escherichia coli based on CRISPR/Cpf1. First, gene knockout and integration efficiency were verified in eight different kinds of protospacer adjacent motif (PAM) regions. Then, the transformation method was optimized, and the efficiency of gene knockout or gene integration of this system increased to nearly 100%, and the large-length fragments could be integrated into the genome in E. coli BL21 (DE3). The system was also optimized by replacing the homologous recombination system in plasmid pEcCpf1, resulting in pEcCpf1H, which could perform precise single-point mutation, terminator insertion, short-sequence insertion, or gene knockout with high efficiency using a 90 nt (nucleotide) single-stranded primer. Further, multiple genes could be edited simultaneously. Next, these two systems were demonstrated in other E. coli strains. Finally, as an application, the system was used to engineer the synthesis pathway of l-histidine in the engineered strain. The titer of l-histidine in a shake flask reached 7.16 g/L, a value increased by 84.1% compared to the starting strain. Thus, this study provided an effective tool for metabolic engineering of E. coli.}, }
@article {pmid35412812, year = {2022}, author = {Scott, H and Sun, D and Beal, J and Kiani, S}, title = {Simulation-Based Engineering of Time-Delayed Safety Switches for Safer Gene Therapies.}, journal = {ACS synthetic biology}, volume = {11}, number = {5}, pages = {1782-1789}, doi = {10.1021/acssynbio.1c00621}, pmid = {35412812}, issn = {2161-5063}, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genetic Therapy ; Humans ; }, abstract = {CRISPR-based gene editing is a powerful tool with great potential for applications in the treatment of many inherited and acquired diseases. The longer that CRISPR gene therapy is maintained within a patient, however, the higher the likelihood that it will result in problematic side effects such as off-target editing or immune response. One approach to mitigating these issues is to link the operation of the therapeutic system to a safety switch that autonomously disables its operation and removes the delivered therapeutics after some amount of time. We present here a simulation-based analysis of the potential for regulating the time delay of such a safety switch using one or two transcriptional regulators and/or recombinases. Combinatorial circuit generation identifies 30 potential architectures for such circuits, which we evaluate in simulation with respect to tunability, sensitivity to parameter values, and sensitivity to cell-to-cell variation. This modeling predicts one of these circuit architectures to have the desired dynamics and robustness, which can be further tested and applied in the context of CRISPR therapeutics.}, }
@article {pmid35338523, year = {2022}, author = {Chen, Y and Jong, TT and Chen, C and Sidransky, E}, title = {CRISPR/Cas9-Based Functional Genomics in Human Induced Pluripotent Stem Cell-Derived Models: Can "the Stars Align" for Neurodegenerative Diseases?.}, journal = {Movement disorders : official journal of the Movement Disorder Society}, volume = {37}, number = {5}, pages = {886-890}, doi = {10.1002/mds.28998}, pmid = {35338523}, issn = {1531-8257}, support = {/HG/NHGRI NIH HHS/United States ; //Intramural Research Program/ ; /NH/NIH HHS/United States ; //The Michael J. Fox Foundation for Parkinson's Research (MJFF)/ ; //Aligning Science Across Parkinson's (ASAP)/ ; /HG/NHGRI NIH HHS/United States ; /NH/NIH HHS/United States ; }, mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; Genomics ; Humans ; *Induced Pluripotent Stem Cells ; *Neurodegenerative Diseases/genetics ; }, }
@article {pmid35598011, year = {2022}, author = {Herrera-Uribe, J and Zaldívar-López, S and Aguilar, C and Entrenas-García, C and Bautista, R and Claros, MG and Garrido, JJ}, title = {Study of microRNA expression in Salmonella Typhimurium-infected porcine ileum reveals miR-194a-5p as an important regulator of the TLR4-mediated inflammatory response.}, journal = {Veterinary research}, volume = {53}, number = {1}, pages = {35}, pmid = {35598011}, issn = {1297-9716}, support = {AGL2014-54089-R//Ministerio de Economía y Competitividad/ ; FPDI-2013-15619//Ministerio de Economía y Competitividad/ ; BES-2012-058642//Ministerio de Economía y Competitividad/ ; AGL2017-87415-R//Ministerio de Economía, Industria y Competitividad, Gobierno de España/ ; IJCI-2017-31382//Ministerio de Economía, Industria y Competitividad, Gobierno de España/ ; }, abstract = {Infection with Salmonella Typhimurium (S. Typhimurium) is a common cause of food-borne zoonosis leading to acute gastroenteritis in humans and pigs, causing economic losses to producers and farmers, and generating a food security risk. In a previous study, we demonstrated that S. Typhimurium infection produces a severe transcriptional activation of inflammatory processes in ileum. However, little is known regarding how microRNAs regulate this response during infection. Here, small RNA sequencing was used to identify 28 miRNAs differentially expressed (DE) in ileum of S. Typhimurium-infected pigs, which potentially regulate 14 target genes involved in immune system processes such as regulation of cytokine production, monocyte chemotaxis, or cellular response to interferon gamma. Using in vitro functional and gain/loss of function (mimics/CRISPR-Cas system) approaches, we show that porcine miR-194a-5p (homologous to human miR-194-5p) regulates TLR4 gene expression, an important molecule involved in pathogen virulence, recognition and activation of innate immunity in Salmonella infection.}, }
@article {pmid35597144, year = {2022}, author = {Wei, Y and Tao, Z and Wan, L and Zong, C and Wu, J and Tan, X and Wang, B and Guo, Z and Zhang, L and Yuan, H and Wang, P and Yang, Z and Wan, Y}, title = {Aptamer-based Cas14a1 biosensor for amplification-free live pathogenic detection.}, journal = {Biosensors &amp; bioelectronics}, volume = {211}, number = {}, pages = {114282}, doi = {10.1016/j.bios.2022.114282}, pmid = {35597144}, issn = {1873-4235}, abstract = {CRISPR-Cas systems have been employed to detect a large variety of pathogenic microorganisms by simply changing the guide RNA sequence. However, these platforms usually rely on nucleic acid extraction and amplification to achieve good sensitivity. Herein, we developed a new platform for the highly specific and sensitive detection of live staphylococcus aureus (S. aureus) based on an Aptamer-based Cas14a1 Biosensor (ACasB), without the need for nucleic acid extraction or amplification. First, the S. aureus specific aptamer was hybrid with a blocker DNA. After the live S. aureus was added, the blocker can be released upon bacteria-aptamer binding. Finally, the released blocker can activate Cas14a1 protein by binding with the sgRNA to generate a change of fluorescent intensity. The ACasB indicates high specificity and sensitivity: it can directly distinguish 400 CFU/ml live S. aureus cells. Comparable to qPCR, the Cas14a1-aptamer biosensor can detect S. aureus with 100% accuracy in complex samples. Therefore, this ACasB for the on-site detection of live S. aureus can broaden its applications in food safety and environmental monitoring.}, }
@article {pmid35595757, year = {2022}, author = {McGaw, C and Garrity, AJ and Munoz, GZ and Haswell, JR and Sengupta, S and Keston-Smith, E and Hunnewell, P and Ornstein, A and Bose, M and Wessells, Q and Jakimo, N and Yan, P and Zhang, H and Alfonse, LE and Ziblat, R and Carte, JM and Lu, WC and Cerchione, D and Hilbert, B and Sothiselvam, S and Yan, WX and Cheng, DR and Scott, DA and DiTommaso, T and Chong, S}, title = {Engineered Cas12i2 is a versatile high-efficiency platform for therapeutic genome editing.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {2833}, pmid = {35595757}, issn = {2041-1723}, abstract = {The CRISPR-Cas type V-I is a family of Cas12i-containing programmable nuclease systems guided by a short crRNA without requirement for a tracrRNA. Here we present an engineered Type V-I CRISPR system (Cas12i), ABR-001, which utilizes a tracr-less guide RNA. The compact Cas12i effector is capable of self-processing pre-crRNA and cleaving dsDNA targets, which facilitates versatile delivery options and multiplexing, respectively. We apply an unbiased mutational scanning approach to enhance initially low editing activity of Cas12i2. The engineered variant, ABR-001, exhibits broad genome editing capability in human cell lines, primary T cells, and CD34+ hematopoietic stem and progenitor cells, with both robust efficiency and high specificity. In addition, ABR-001 achieves a high level of genome editing when delivered via AAV vector to HEK293T cells. This work establishes ABR-001 as a versatile, specific, and high-performance platform for ex vivo and in vivo gene therapy.}, }
@article {pmid35595728, year = {2022}, author = {Schwartz, EA and McBride, TM and Bravo, JPK and Wrapp, D and Fineran, PC and Fagerlund, RD and Taylor, DW}, title = {Structural rearrangements allow nucleic acid discrimination by type I-D Cascade.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {2829}, pmid = {35595728}, issn = {2041-1723}, support = {R35GM138348//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; }, abstract = {CRISPR-Cas systems are adaptive immune systems that protect prokaryotes from foreign nucleic acids, such as bacteriophages. Two of the most prevalent CRISPR-Cas systems include type I and type III. Interestingly, the type I-D interference proteins contain characteristic features of both type I and type III systems. Here, we present the structures of type I-D Cascade bound to both a double-stranded (ds)DNA and a single-stranded (ss)RNA target at 2.9 and 3.1 Å, respectively. We show that type I-D Cascade is capable of specifically binding ssRNA and reveal how PAM recognition of dsDNA targets initiates long-range structural rearrangements that likely primes Cas10d for Cas3' binding and subsequent non-target strand DNA cleavage. These structures allow us to model how binding of the anti-CRISPR protein AcrID1 likely blocks target dsDNA binding via competitive inhibition of the DNA substrate engagement with the Cas10d active site. This work elucidates the unique mechanisms used by type I-D Cascade for discrimination of single-stranded and double stranded targets. Thus, our data supports a model for the hybrid nature of this complex with features of type III and type I systems.}, }
@article {pmid35595574, year = {2022}, author = {Li, L and Shen, G and Wu, M and Jiang, J and Xia, Q and Lin, P}, title = {CRISPR-Cas-mediated diagnostics.}, journal = {Trends in biotechnology}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.tibtech.2022.04.006}, pmid = {35595574}, issn = {1879-3096}, abstract = {An ideal molecular diagnostic method should be sensitive, specific, low cost, rapid, portable, and easy to operate. Traditional nucleic acid detection methods based mainly on PCR technology have not only high sensitivity and specificity, but also some limitations, such as the need for expensive equipment and skilled technicians, being both time and labor intensive, and difficult to implement in some regions. However, with the continuous development of CRISPR-Cas technology and its application in molecular diagnosis, new approaches have been used for the construction of molecular diagnostic systems. In this review, we discuss recent advances in CRISPR-based molecular diagnostic technologies and highlight the revolution they bring to the field of molecular diagnostics.}, }
@article {pmid35594718, year = {2022}, author = {Nath, A and Bhattacharjee, R and Nandi, A and Sinha, A and Kar, S and Manoharan, N and Mitra, S and Mojumdar, A and Panda, PK and Patro, S and Dutt, A and Ahuja, R and Verma, SK and Suar, M}, title = {Phage delivered CRISPR-Cas system to combat multidrug-resistant pathogens in gut microbiome.}, journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie}, volume = {151}, number = {}, pages = {113122}, doi = {10.1016/j.biopha.2022.113122}, pmid = {35594718}, issn = {1950-6007}, abstract = {The Host-microbiome interactions that exist inside the gut microbiota operate in a synergistic and abnormal manner. Additionally, the normal homeostasis and functioning of gut microbiota are frequently disrupted by the intervention of Multi-Drug Resistant (MDR) pathogens. CRISPR-Cas (CRISPR-associated protein with clustered regularly interspersed short palindromic repeats) recognized as a prokaryotic immune system has emerged as an effective genome-editing tool to edit and delete specific microbial genes for the expulsion of bacteria through bactericidal action. In this review, we demonstrate many functioning CRISPR-Cas systems against the anti-microbial resistance of multiple pathogens, which infiltrate the gastrointestinal tract. Moreover, we discuss the advancement in the development of a phage-delivered CRISPR-Cas system for killing a gut MDR pathogen. We also discuss a combinatorial approach to use bacteriophage as a delivery system for the CRISPR-Cas gene for targeting a pathogenic community in the gut microbiome to resensitize the drug sensitivity. Finally, we discuss engineered phage as a plausible potential option for the CRISPR-Cas system for pathogenic killing and improvement of the efficacy of the system.}, }
@article {pmid35592579, year = {2022}, author = {Chen, H and Neubauer, M and Wang, JP}, title = {Enhancing HR Frequency for Precise Genome Editing in Plants.}, journal = {Frontiers in plant science}, volume = {13}, number = {}, pages = {883421}, pmid = {35592579}, issn = {1664-462X}, abstract = {Gene-editing tools, such as Zinc-fingers, TALENs, and CRISPR-Cas, have fostered a new frontier in the genetic improvement of plants across the tree of life. In eukaryotes, genome editing occurs primarily through two DNA repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is the primary mechanism in higher plants, but it is unpredictable and often results in undesired mutations, frameshift insertions, and deletions. Homology-directed repair (HDR), which proceeds through HR, is typically the preferred editing method by genetic engineers. HR-mediated gene editing can enable error-free editing by incorporating a sequence provided by a donor template. However, the low frequency of native HR in plants is a barrier to attaining efficient plant genome engineering. This review summarizes various strategies implemented to increase the frequency of HDR in plant cells. Such strategies include methods for targeting double-strand DNA breaks, optimizing donor sequences, altering plant DNA repair machinery, and environmental factors shown to influence HR frequency in plants. Through the use and further refinement of these methods, HR-based gene editing may one day be commonplace in plants, as it is in other systems.}, }
@article {pmid35589712, year = {2022}, author = {Philippe, C and Morency, C and Plante, PL and Zufferey, E and Achigar, R and Tremblay, DM and Rousseau, GM and Goulet, A and Moineau, S}, title = {A truncated anti-CRISPR protein prevents spacer acquisition but not interference.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {2802}, pmid = {35589712}, issn = {2041-1723}, abstract = {CRISPR-Cas systems in prokaryotic cells provide an adaptive immunity against invading nucleic acids. For example, phage infection leads to addition of new immunity (spacer acquisition) and DNA cleavage (interference) in the bacterial model species Streptococcus thermophilus, which primarily relies on Cas9-containing CRISPR-Cas systems. Phages can counteract this defense system through mutations in the targeted protospacers or by encoding anti-CRISPR proteins (ACRs) that block Cas9 interference activity. Here, we show that S. thermophilus can block ACR-containing phages when the CRISPR immunity specifically targets the acr gene. This in turn selects for phage mutants carrying a deletion within the acr gene. Remarkably, a truncated acrIIA allele, found in a wild-type virulent streptococcal phage, does not block the interference activity of Cas9 but still prevents the acquisition of new immunities, thereby providing an example of an ACR specifically inhibiting spacer acquisition.}, }
@article {pmid35585651, year = {2022}, author = {Osteikoetxea, X and Silva, A and Lázaro-Ibáñez, E and Salmond, N and Shatnyeva, O and Stein, J and Schick, J and Wren, S and Lindgren, J and Firth, M and Madsen, A and Mayr, LM and Overman, R and Davies, R and Dekker, N}, title = {Engineered Cas9 extracellular vesicles as a novel gene editing tool.}, journal = {Journal of extracellular vesicles}, volume = {11}, number = {5}, pages = {e12225}, doi = {10.1002/jev2.12225}, pmid = {35585651}, issn = {2001-3078}, support = {739593//European Union's Horizon 2020 Research and Innovation Programme/ ; }, mesh = {CRISPR-Cas Systems/genetics ; *Extracellular Vesicles ; *Gene Editing ; HEK293 Cells ; Humans ; Proprotein Convertase 9/genetics ; }, abstract = {Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic applications require efficient cargo loading. Here, we developed new methods for CRISPR/Cas9 loading into EVs through reversible heterodimerization of Cas9-fusions with EV sorting partners. Cas9-loaded EVs were collected from engineered Expi293F cells using standard methodology, characterized using nanoparticle tracking analysis, western blotting, and transmission electron microscopy and analysed for CRISPR/Cas9-mediated functional gene editing in a Cre-reporter cellular assay. Light-induced dimerization using Cryptochrome 2 combined with CD9 or a Myristoylation-Palmitoylation-Palmitoylation lipid modification resulted in efficient loading with approximately 25 Cas9 molecules per EV and high functional delivery with 51% gene editing of the Cre reporter cassette in HEK293 and 25% in HepG2 cells, respectively. This approach was also effective for targeting knock-down of the therapeutically relevant PCSK9 gene with 6% indel efficiency in HEK293. Cas9 transfer was detergent-sensitive and associated with the EV fractions after size exclusion chromatography, indicative of EV-mediated transfer. Considering the advantages of EVs over other delivery vectors we envision that this study will prove useful for a range of therapeutic applications, including CRISPR/Cas9 mediated genome editing.}, }
@article {pmid35584220, year = {2022}, author = {Fan, N and Bian, X and Li, M and Chen, J and Wu, H and Peng, Q and Bai, H and Cheng, W and Kong, L and Ding, S and Li, S and Cheng, W}, title = {Hierarchical self-uncloaking CRISPR-Cas13a-customized RNA nanococoons for spatial-controlled genome editing and precise cancer therapy.}, journal = {Science advances}, volume = {8}, number = {20}, pages = {eabn7382}, doi = {10.1126/sciadv.abn7382}, pmid = {35584220}, issn = {2375-2548}, mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; *Neoplasms/genetics/therapy ; RNA ; RNA, Messenger/genetics ; }, abstract = {CRISPR-Cas13a holds enormous potential for developing precise RNA editing. However, spatial manipulation of CRISPR-Cas13a activity remains a daunting challenge for elaborately regulating localized RNase function. Here, we designed hierarchical self-uncloaking CRISPR-Cas13a-customized RNA nanococoons (RNCOs-D), featuring tumor-specific recognition and spatial-controlled activation of Cas13a, for precise cancer synergistic therapy. RNCOs-D consists of programmable RNA nanosponges (RNSs) capable of targeted delivery and caging chemotherapeutic drug, and nanocapsules (NCs) anchored on RNSs for cloaking Cas13a/crRNA ribonucleoprotein (Cas13a RNP) activity. The acidic endo/lysosomal microenvironment stimulates the outer decomposition of NCs with concomitant Cas13a RNP activity revitalization, while the inner disassembly through trans-cleavage of RNSs initiated by cis-recognition and cleavage of EGFR variant III (EGFRvIII) mRNA. RNCOs-D demonstrates the effective EGFRvIII mRNA silencing for synergistic therapy of glioblastoma cancer cells in vitro and in vivo. The engineering of RNSs, together with efficient Cas13a activity regulation, holds immense prospect for multimodal and synergistic cancer therapy.}, }
@article {pmid35583740, year = {2022}, author = {Wen, Z and Qian, F and Zhang, J and Jiang, Y and Yang, S}, title = {Genome Editing of Corynebacterium glutamicum Using CRISPR-Cpf1 System.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2479}, number = {}, pages = {189-206}, pmid = {35583740}, issn = {1940-6029}, mesh = {CRISPR-Cas Systems/genetics ; *Corynebacterium glutamicum/genetics ; *Gene Editing/methods ; }, abstract = {Corynebacterium glutamicum, as an important microbial chassis, has great potential in industrial application. However, complicated genetic modification is severely slowed by lack of efficient genome editing tools. The Streptococcus pyogenes (Sp) CRISPR-Cas9 system has been verified as a very powerful tool for mediating genome alteration in many microorganisms but cannot work well in C. glutamicum. We recently developed two Francisella novicida (Fn) CRISPR-Cpf1 assisted systems for genome editing via homologous recombination in C. glutamicum. Here, we describe the protocols and demonstrated that N iterative rounds of genome editing can be achieved in 3 N + 4 or 3 N + 2 days, respectively.}, }
@article {pmid35583739, year = {2022}, author = {Hong, W and Zhang, J and Cui, G and Zhou, Q and Wang, P and Wang, Y}, title = {Highly Efficient Genome Editing in Clostridium difficile Using the CRISPR-Cpf1 System.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2479}, number = {}, pages = {175-187}, pmid = {35583739}, issn = {1940-6029}, mesh = {CRISPR-Cas Systems/genetics ; *Clostridioides difficile/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing/methods ; Plasmids/genetics ; }, abstract = {Clostridium difficile is often the primary cause of nosocomial diarrhea, leading to thousands of deaths annually worldwide. The availability of an efficient genome editing tool for C. difficile is essential to understanding its pathogenic mechanism and physiological behavior. Here, we describe a streamlined CRISPR-Cpf1-based protocol to achieve precise genome editing in C. difficile with high efficiencies. Our work highlighted the first application of CRISPR-Cpf1 for genome editing in C. difficile, which are both crucial for understanding pathogenic mechanism of C. difficile and developing strategies to fight against C. difficile infection (CDI). In addition, for the DNA cloning, we developed a one-step-assembly protocol along with a Python-based algorithm for automatic primer design, shortening the time for plasmid construction to half that of conventional procedures. Approaches we developed herein are easily and broadly applicable to other microorganisms. Our results provide valuable guidance for establishing CRISPR-Cpf1 as a versatile genome engineering tool in prokaryotic cells.}, }
@article {pmid35583738, year = {2022}, author = {Wozniak, KJ and Simmons, LA}, title = {Genome Editing Methods for Bacillus subtilis.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2479}, number = {}, pages = {159-174}, pmid = {35583738}, issn = {1940-6029}, mesh = {*Bacillus subtilis/genetics ; Bacterial Proteins/genetics ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genetic Engineering ; Humans ; }, abstract = {Bacillus subtilis is a widely studied Gram-positive bacterium that serves as an important model for understanding processes critical for several areas of biology including biotechnology and human health. B. subtilis has several advantages as a model organism: it is easily grown under laboratory conditions, it has a rapid doubling time, it is relatively inexpensive to maintain, and it is nonpathogenic. Over the last 50 years, advancements in genetic engineering have continued to make B. subtilis a genetic workhorse in scientific discovery. In this chapter, we describe methods for traditional gene disruptions, use of gene deletion libraries from the Bacillus Genetic Stock Center, allelic exchange, CRISPRi, and CRISPR/Cas9. Additionally, we provide general materials and equipment needed, strengths and limitations, time considerations, and troubleshooting notes to perform each method. Use of the methods outlined in this chapter will allow researchers to create gene insertions, deletions, substitutions, and RNA interference strains through a variety of methods custom to each application.}, }
@article {pmid35583737, year = {2022}, author = {Penewit, K and Salipante, SJ}, title = {Recombineering in Staphylococcus aureus.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2479}, number = {}, pages = {135-157}, pmid = {35583737}, issn = {1940-6029}, mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genetic Engineering ; Humans ; Recombinases/genetics ; *Staphylococcal Infections/genetics ; *Staphylococcus aureus/genetics ; }, abstract = {Recombineering has proven to be an extraordinarily powerful and versatile approach for the modification of bacterial genomes, but has historically not been possible in the important opportunistic pathogen Staphylococcus aureus. After evaluating the activity of various recombinases in S. aureus, we developed methods for recombineering in that organism using synthetic, single-stranded DNA oligonucleotides. This approach can be coupled to CRISPR/Cas9-mediated lethal counterselection in order to improve the efficiency with which recombinant S. aureus are recovered, which is especially useful in instances where mutants lack a selectable phenotype. These methods provide a rapid, scalable, precise, and inexpensive means to engineer point mutations, variable-length deletions, and short insertions into the S. aureus genome.}, }
@article {pmid35583736, year = {2022}, author = {Ellington, AJ and Reisch, CR}, title = {Generating Single Nucleotide Point Mutations in E. coli with the No-SCAR System.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2479}, number = {}, pages = {119-133}, pmid = {35583736}, issn = {1940-6029}, mesh = {*CRISPR-Cas Systems ; *Escherichia coli/genetics ; Gene Editing/methods ; Nucleotides ; Point Mutation ; }, abstract = {Genetic manipulation of microbial genomes is highly relevant for studying biological systems and the development of biotechnologies. In E. coli, λ-Red recombineering is one of the most widely used gene-editing methods, enabling site-specific insertions, deletions, and point mutations of any genomic locus. The no-SCAR system combines λ-Red recombineering with CRISPR/Cas9 for programmable selection of recombinant cells. Recombineering results in the transient production of heteroduplex DNA, as only one strand of DNA is initially altered, leaving the mismatched bases susceptible to repair by the host methyl-directed mismatch repair (MMR) system and reduces the efficiency of generating single nucleotide point mutations. Here we describe a method, where expression of cas9 and the MMR-inhibiting mutLE32K variant are independently controlled by anhydrotetracycline- and cumate-inducible promoters from the pCas9CyMutL plasmid. Thus, MMR is selectively inhibited until recombinant cells have undergone replication and the desired mutation is permanently incorporated. By transiently inhibiting MMR, the accumulation of off-target mutations typically associated with MMR-deficient cell types is minimized. Methods for designing the editing template and sgRNA, cloning of the sgRNA, induction of λ-Red and MutLE32K, the transformation of editing oligo, and induction of Cas9 for mutant selection are detailed within.}, }
@article {pmid35583735, year = {2022}, author = {Wang, Z and Wang, Y and Ji, Q}, title = {Genome Editing in Klebsiella pneumoniae Using CRISPR/Cas9 Technology.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2479}, number = {}, pages = {105-117}, pmid = {35583735}, issn = {1940-6029}, mesh = {CRISPR-Cas Systems/genetics ; Cytidine Deaminase/genetics ; *Gene Editing/methods ; *Klebsiella pneumoniae/genetics/metabolism ; Technology ; }, abstract = {CRISPR/Cas9 systems have been widely adopted for genetic manipulation in diverse biological systems owing to the ease of use and high efficiency. We have recently developed a CRISPR/Cas9-based genome editing system (pCasKP-pSGKP) by coupling a CRISPR/Cas9 system with the lambda Red recombination system as well as a cytidine deaminase-mediated base editing system (pBECKP) in Klebsiella pneumoniae, enabling rapid, scarless, and efficient genetic manipulation in diverse K. pneumoniae strains. In this chapter, we introduce the detailed procedures of using these two tools for genome editing in K. pneumoniae.}, }
@article {pmid35395152, year = {2022}, author = {Hobbs, SJ and Wein, T and Lu, A and Morehouse, BR and Schnabel, J and Leavitt, A and Yirmiya, E and Sorek, R and Kranzusch, PJ}, title = {Phage anti-CBASS and anti-Pycsar nucleases subvert bacterial immunity.}, journal = {Nature}, volume = {605}, number = {7910}, pages = {522-526}, pmid = {35395152}, issn = {1476-4687}, mesh = {Bacteria/metabolism ; Bacterial Proteins/metabolism ; Bacteriophage T4/metabolism ; *Bacteriophages/physiology ; CRISPR-Cas Systems/genetics ; Endonucleases/metabolism ; Escherichia coli/metabolism ; Nucleotides, Cyclic/metabolism ; Oligonucleotides ; Pyrimidines/metabolism ; }, abstract = {The cyclic oligonucleotide-based antiphage signalling system (CBASS) and the pyrimidine cyclase system for antiphage resistance (Pycsar) are antiphage defence systems in diverse bacteria that use cyclic nucleotide signals to induce cell death and prevent viral propagation1,2. Phages use several strategies to defeat host CRISPR and restriction-modification systems3-10, but no mechanisms are known to evade CBASS and Pycsar immunity. Here we show that phages encode anti-CBASS (Acb) and anti-Pycsar (Apyc) proteins that counteract defence by specifically degrading cyclic nucleotide signals that activate host immunity. Using a biochemical screen of 57 phages in Escherichia coli and Bacillus subtilis, we discover Acb1 from phage T4 and Apyc1 from phage SBSphiJ as founding members of distinct families of immune evasion proteins. Crystal structures of Acb1 in complex with 3'3'-cyclic GMP-AMP define a mechanism of metal-independent hydrolysis 3' of adenosine bases, enabling broad recognition and degradation of cyclic dinucleotide and trinucleotide CBASS signals. Structures of Apyc1 reveal a metal-dependent cyclic NMP phosphodiesterase that uses relaxed specificity to target Pycsar cyclic pyrimidine mononucleotide signals. We show that Acb1 and Apyc1 block downstream effector activation and protect from CBASS and Pycsar defence in vivo. Active Acb1 and Apyc1 enzymes are conserved in phylogenetically diverse phages, demonstrating that cleavage of host cyclic nucleotide signals is a key strategy of immune evasion in phage biology.}, }
@article {pmid35587321, year = {2022}, author = {Kumar, G and Jagadeeshwari, U and Sreya, P and Shabbir, A and Sasikala, C and Ramana, CV}, title = {A genomic overview including polyphasic taxonomy of Thalassoroseus pseudoceratinae gen. nov., sp. nov. isolated from a marine sponge, Pseudoceratina sp.}, journal = {Antonie van Leeuwenhoek}, volume = {}, number = {}, pages = {}, pmid = {35587321}, issn = {1572-9699}, abstract = {A pink-coloured, salt- and alkali-tolerant planctomycetal strain (JC658T) with oval to pear-shaped, motile, aerobic, Gram-negative stained cells was isolated from a marine sponge, Pseudoceratina sp. Strain JC658T shares the highest 16S rRNA gene sequence identity with Maioricimonas rarisocia Mal4T (< 89.2%) in the family Planctomycetaceae. The genomic analysis of the new strain indicates its biotechnological potential for the production of various industrially important enzymes, notably sulfatases and carbohydrate-active enzymes (CAZymes), and also potential antimicrobial compounds. Several genes encoding restriction-modification (RM) and CRISPR-CAS systems are also present. NaCl is obligate for growth, of which strain JC658T can tolerate a concentration up to 6% (w/v). Optimum pH and temperature for growth are 8.0 (range 7.0-9.0) and 25 ºC (range 10-40 °C), respectively. The major respiratory quinone of strain JC658T is MK6. Major fatty acids are C16:1ω7c/C16:1ω6c, C18:0 and C16:0. Major polar lipids are phosphatidylcholine, phosphatidyl-dimethylethanolamine and phosphatidyl-monomethylethanolamine. The genomic size of strain JC658T is 7.36 Mb with a DNA G + C content of 54.6 mol%. Based on phylogenetic, genomic (ANI, AAI, POCP, dDDH), chemotaxonomic, physiological and biochemical characteristics, we conclude that strain JC658T belongs to a novel genus and constitutes a novel species within the family Planctomycetaceae, for which we propose the name Thalassoroseus pseudoceratinae gen. nov., sp. nov. The novel species is represented by the type strain JC658T (= KCTC 72881 T = NBRC 114371 T).}, }
@article {pmid35587292, year = {2022}, author = {Van Vu, T and Das, S and Hensel, G and Kim, JY}, title = {Genome editing and beyond: what does it mean for the future of plant breeding?.}, journal = {Planta}, volume = {255}, number = {6}, pages = {130}, pmid = {35587292}, issn = {1432-2048}, support = {2020R1I1A1A01072130//National Research Foundation of Korea/ ; 2020M3A9I4038352//National Research Foundation of Korea/ ; 2020R1A6A1A03044344//National Research Foundation of Korea/ ; Germany's Excellence Strategy - EXC-2048/1 - project ID 390686111//Deutsche Forschungsgemeinschaft/ ; 426557363//Deutsche Forschungsgemeinschaft/ ; 458717903//Deutsche Forschungsgemeinschaft/ ; ZS/2018/06/93171//European Regional Development Fund/ ; CZ.02.1.01./0.0/0.0/16_019/0000827//Czech Science Foundation/ ; SPP 813103381//Czech Science Foundation/ ; }, abstract = {MAIN CONCLUSION: Genome editing offers revolutionized solutions for plant breeding to sustain food production to feed the world by 2050. Therefore, genome-edited products are increasingly recognized via more relaxed legislation and community adoption. The world population and food production are disproportionally growing in a manner that would have never matched each other under the current agricultural practices. The emerging crisis is more evident with the subtle changes in climate and the running-off of natural genetic resources that could be easily used in breeding in conventional ways. Under these circumstances, affordable CRISPR-Cas-based gene-editing technologies have brought hope and charged the old plant breeding machine with the most energetic and powerful fuel to address the challenges involved in feeding the world. What makes CRISPR-Cas the most powerful gene-editing technology? What are the differences between it and the other genetic engineering/breeding techniques? Would its products be labeled as "conventional" or "GMO"? There are so many questions to be answered, or that cannot be answered within the limitations of our current understanding. Therefore, we would like to discuss and answer some of the mentioned questions regarding recent progress in technology development. We hope this review will offer another view on the role of CRISPR-Cas technology in future of plant breeding for food production and beyond.}, }
@article {pmid35586709, year = {2022}, author = {Yin, T and Luo, J and Huang, D and Li, H}, title = {Current Progress of Mitochondrial Genome Editing by CRISPR.}, journal = {Frontiers in physiology}, volume = {13}, number = {}, pages = {883459}, doi = {10.3389/fphys.2022.883459}, pmid = {35586709}, issn = {1664-042X}, }
@article {pmid35581343, year = {2022}, author = {Perdigoto, CN}, title = {TALEDs complete the toolkit for editing human mitochondrial DNA.}, journal = {Nature structural &amp; molecular biology}, volume = {29}, number = {5}, pages = {415}, doi = {10.1038/s41594-022-00781-z}, pmid = {35581343}, issn = {1545-9985}, mesh = {CRISPR-Cas Systems/genetics ; *DNA, Mitochondrial/genetics ; *Gene Editing ; Humans ; Mitochondria/genetics ; }, }
@article {pmid35573771, year = {2022}, author = {Wu, X and Wang, S and Li, C and Shi, J and Peng, Z and Liu, C and Han, H and Ma, Y and Zheng, L and Xu, S and Du, W and Li, J and Zhang, F}, title = {CRISPR/Cas9-Mediated Knockout of the Dicer and Ago2 Genes in BHK-21 Cell Promoted Seneca Virus A Replication and Enhanced Autophagy.}, journal = {Frontiers in cellular and infection microbiology}, volume = {12}, number = {}, pages = {865744}, pmid = {35573771}, issn = {2235-2988}, mesh = {Animals ; Autophagy ; *CRISPR-Cas Systems ; DNA Viruses ; Female ; *Picornaviridae/genetics ; RNA Interference ; Swine ; Virus Replication ; }, abstract = {RNA interference (RNAi) is a major form of antiviral defense in host cells, and Ago2 and Dicer are the major proteins of RNAi. The Senecavirus A (SVA) is a reemerging virus, resulting in vesicular lesions in sows and a sharp decline in neonatal piglet production. In this study, CRISPR/Cas9 technology was used to knock out Ago2 and Dicer genes in BHK-21 cell lines used for SVA vaccine production. Cell clones with homozygous frameshift mutations of Ago2 and Dicer genes were successfully identified. The two knockout cell lines were named BHK-DicerΔ- and BHK-Ago2Δ-. Results showed that the two genes' knockout cell lines were capable of stable passage and the cell growth rate did not change significantly. The replication rate and virus titers of SVA were significantly increased in knockout cell lines, indicating that RNAi could inhibit SVA replication. In addition, compared with normal cells, autophagy was significantly enhanced after SVA-infected knockout cell lines, while there was no significant difference in autophagy between the knockout and normal cell lines without SVA. The results confirmed that SVA could enhance the autophagy in knockout cells and promote viral replication. The two knockout cell lines can obtain viruses with high viral titers and have good application prospects in the production of SVA vaccine. At the same time, the RNAi knockout cell lines provide convenience for further studies on RNAi and SVA resistance to RNAi, and it lays a foundation for further study of SVA infection characteristics and screening of new therapeutic drugs and drug targets.}, }
@article {pmid35559673, year = {2022}, author = {Feng, X and Tang, M and Dede, M and Su, D and Pei, G and Jiang, D and Wang, C and Chen, Z and Li, M and Nie, L and Xiong, Y and Li, S and Park, JM and Zhang, H and Huang, M and Szymonowicz, K and Zhao, Z and Hart, T and Chen, J}, title = {Genome-wide CRISPR screens using isogenic cells reveal vulnerabilities conferred by loss of tumor suppressors.}, journal = {Science advances}, volume = {8}, number = {19}, pages = {eabm6638}, doi = {10.1126/sciadv.abm6638}, pmid = {35559673}, issn = {2375-2548}, mesh = {*Antineoplastic Agents ; CRISPR-Cas Systems ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genes, Tumor Suppressor ; Humans ; *Neoplasms/genetics ; Synthetic Lethal Mutations ; }, abstract = {Exploiting cancer vulnerabilities is critical for the discovery of anticancer drugs. However, tumor suppressors cannot be directly targeted because of their loss of function. To uncover specific vulnerabilities for cells with deficiency in any given tumor suppressor(s), we performed genome-scale CRISPR loss-of-function screens using a panel of isogenic knockout cells we generated for 12 common tumor suppressors. Here, we provide a comprehensive and comparative dataset for genetic interactions between the whole-genome protein-coding genes and a panel of tumor suppressor genes, which allows us to uncover known and new high-confidence synthetic lethal interactions. Mining this dataset, we uncover essential paralog gene pairs, which could be a common mechanism for interpreting synthetic lethality. Moreover, we propose that some tumor suppressors could be targeted to suppress proliferation of cells with deficiency in other tumor suppressors. This dataset provides valuable information that can be further exploited for targeted cancer therapy.}, }
@article {pmid35534557, year = {2022}, author = {Eisenstein, M}, title = {Base editing marches on the clinic.}, journal = {Nature biotechnology}, volume = {40}, number = {5}, pages = {623-625}, doi = {10.1038/s41587-022-01326-x}, pmid = {35534557}, issn = {1546-1696}, mesh = {Ambulatory Care Facilities ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; }, }
@article {pmid35436106, year = {2022}, author = {Park, H and Osman, EA and Cromwell, CR and St Laurent, CD and Liu, Y and Kitova, EN and Klassen, JS and Hubbard, BP and Macauley, MS and Gibbs, JM}, title = {CRISPR-Click Enables Dual-Gene Editing with Modular Synthetic sgRNAs.}, journal = {Bioconjugate chemistry}, volume = {33}, number = {5}, pages = {858-868}, doi = {10.1021/acs.bioconjchem.2c00106}, pmid = {35436106}, issn = {1520-4812}, mesh = {Alkynes ; Azides/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; RNA, Guide/genetics/metabolism ; }, abstract = {Gene-editing systems such as CRISPR-Cas9 readily enable individual gene phenotypes to be studied through loss of function. However, in certain instances, gene compensation can obfuscate the results of these studies, necessitating the editing of multiple genes to properly identify biological pathways and protein function. Performing multiple genetic modifications in cells remains difficult due to the requirement for multiple rounds of gene editing. While fluorescently labeled guide RNAs (gRNAs) are routinely used in laboratories for targeting CRISPR-Cas9 to disrupt individual loci, technical limitations in single gRNA (sgRNA) synthesis hinder the expansion of this approach to multicolor cell sorting. Here, we describe a modular strategy for synthesizing sgRNAs where each target sequence is conjugated to a unique fluorescent label, which enables fluorescence-activated cell sorting (FACS) to isolate cells that incorporate the desired combination of gene-editing constructs. We demonstrate that three short strands of RNA functionalized with strategically placed 5'-azide and 3'-alkyne terminal deoxyribonucleotides can be assembled in a one-step, template-assisted, copper-catalyzed alkyne-azide cycloaddition to generate fully functional, fluorophore-modified sgRNAs. Using these synthetic sgRNAs in combination with FACS, we achieved selective cleavage of two targeted genes, either separately as a single-color experiment or in combination as a dual-color experiment. These data indicate that our strategy for generating double-clicked sgRNA allows for Cas9 activity in cells. By minimizing the size of each RNA fragment to 41 nucleotides or less, this strategy is well suited for custom, scalable synthesis of sgRNAs.}, }
@article {pmid35427721, year = {2022}, author = {Avci-Adali, M and A Santos, H}, title = {Current trends in delivery of non-viral nucleic acid-based therapeutics for improved efficacy.}, journal = {Advanced drug delivery reviews}, volume = {185}, number = {}, pages = {114297}, doi = {10.1016/j.addr.2022.114297}, pmid = {35427721}, issn = {1872-8294}, mesh = {CRISPR-Cas Systems ; Gene Editing ; Humans ; Nanomedicine ; *Nucleic Acids ; }, }
@article {pmid35422459, year = {2022}, author = {}, title = {Forum: CRISPR screening roundtable with Stegmaier and Doench.}, journal = {Nature biotechnology}, volume = {40}, number = {5}, pages = {655}, doi = {10.1038/s41587-022-01303-4}, pmid = {35422459}, issn = {1546-1696}, mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; *Mass Screening ; Research ; }, }
@article {pmid35350865, year = {2022}, author = {Wasala, NB and Million, ED and Watkins, TB and Wasala, LP and Han, J and Yue, Y and Lu, B and Chen, SJ and Hakim, CH and Duan, D}, title = {The gRNA Vector Level Determines the Outcome of Systemic AAV CRISPR Therapy for Duchenne Muscular Dystrophy.}, journal = {Human gene therapy}, volume = {33}, number = {9-10}, pages = {518-528}, doi = {10.1089/hum.2021.130}, pmid = {35350865}, issn = {1557-7422}, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Dependovirus/genetics/metabolism ; *Dystrophin/genetics/metabolism ; Gene Editing/methods ; Genetic Therapy/methods ; Mice ; Mice, Inbred mdx ; Muscle, Skeletal/metabolism ; *Muscular Dystrophy, Duchenne/genetics/therapy ; RNA, Guide/genetics/metabolism ; }, abstract = {Adeno-associated virus (AAV)-mediated clustered regularly interspaced short palindromic repeats (CRISPR) editing holds promise to restore missing dystrophin in Duchenne muscular dystrophy (DMD). Intramuscular coinjection of CRISPR-associated protein 9 (Cas9) and guide RNA (gRNA) vectors resulted in robust dystrophin restoration in short-term studies in the mdx mouse model of DMD. Intriguingly, this strategy failed to yield efficient dystrophin rescue in muscle in a long-term (18-month) systemic injection study. In-depth analyses revealed a selective loss of the gRNA vector after long-term systemic, but not short-term local injection. To determine whether preferential gRNA vector depletion is due to the mode of delivery (local vs. systemic) or the duration of the study (short term vs. long term), we conducted a short-term systemic injection study. The gRNA (4e12 vg/mouse in the 1:1 group or 1.2e13 vg/mouse in the 3:1 group) and Cas9 (4e12 vg/mouse) vectors were coinjected intravenously into 4-week-old mdx mice. The ratio of the gRNA to Cas9 vector genome copy dropped from 1:1 and 3:1 at injection to 0.4:1 and 1:1 at harvest 3 months later, suggesting that the route of administration, rather than the experimental duration, determines preferential gRNA vector loss. Consistent with our long-term systemic injection study, the vector ratio did not influence Cas9 expression. However, the 3:1 group showed significantly higher dystrophin expression and genome editing, better myofiber size distribution, and a more pronounced improvement in muscle function and electrocardiography. Our data suggest that the gRNA vector dose determines the outcome of systemic AAV CRISPR therapy for DMD.}, }
@article {pmid34989166, year = {2022}, author = {Zhang, C and Ren, H and Liu, G and Li, J and Wang, X and Zhang, Y}, title = {Effective Genome Editing Using CRISPR-Cas9 Nanoflowers.}, journal = {Advanced healthcare materials}, volume = {11}, number = {10}, pages = {e2102365}, doi = {10.1002/adhm.202102365}, pmid = {34989166}, issn = {2192-2659}, support = {2021YFC2102300//National Key Research and Development Program/ ; 32071384//National Natural Science Foundation of China/ ; //One-thousand Young Talent Program of China/ ; }, mesh = {Animals ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Mice ; Micelles ; *Nanoparticles ; Polymers/metabolism ; }, abstract = {CRISPR-Cas9 as a powerful gene-editing tool has tremendous potential for the treatment of genetic diseases. Herein, a new mesoporous nanoflower (NF)-like delivery nanoplatform termed Cas9-NF is reported by crosslinking Cas9 and polymeric micelles that enables efficient intracellular delivery and controlled release of Cas9 in response to reductive microenvironment in tumor cells. The flower morphology is flexibly tunable by the protein concentration and different types of crosslinkers. Cas9 protein, embedded between polymeric micelles and protected by Cas9-NF, remains stable even under extreme pH conditions. Responsive cleavage of crosslinkers in tumor cells, leads to the traceless release of Cas9 for efficient gene knockout in nucleus. This crosslinked nanoparticle exhibits excellent capability of downregulating oncogene expression and inhibiting tumor growth in a murine tumor model. Taken together, these findings pave a new pathway toward the application of the protein-micelle crosslinked nanoflower for protein delivery, which warrants further investigations for gene regulation and cancer treatment.}, }
@article {pmid34887556, year = {2022}, author = {Anzalone, AV and Gao, XD and Podracky, CJ and Nelson, AT and Koblan, LW and Raguram, A and Levy, JM and Mercer, JAM and Liu, DR}, title = {Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing.}, journal = {Nature biotechnology}, volume = {40}, number = {5}, pages = {731-740}, pmid = {34887556}, issn = {1546-1696}, support = {R01 HL156647/HL/NHLBI NIH HHS/United States ; RM1 HG00949//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; Liu investigatorship//Howard Hughes Medical Institute (HHMI)/ ; U01 AI142756/AI/NIAID NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; }, mesh = {Base Sequence ; *CRISPR-Cas Systems ; Chromosome Inversion ; DNA/genetics ; *Gene Editing/methods ; Humans ; RNA, Guide/genetics ; }, abstract = {The targeted deletion, replacement, integration or inversion of genomic sequences could be used to study or treat human genetic diseases, but existing methods typically require double-strand DNA breaks (DSBs) that lead to undesired consequences, including uncontrolled indel mixtures and chromosomal abnormalities. Here we describe twin prime editing (twinPE), a DSB-independent method that uses a prime editor protein and two prime editing guide RNAs (pegRNAs) for the programmable replacement or excision of DNA sequences at endogenous human genomic sites. The two pegRNAs template the synthesis of complementary DNA flaps on opposing strands of genomic DNA, which replace the endogenous DNA sequence between the prime-editor-induced nick sites. When combined with a site-specific serine recombinase, twinPE enabled targeted integration of gene-sized DNA plasmids (>5,000 bp) and targeted sequence inversions of 40 kb in human cells. TwinPE expands the capabilities of precision gene editing and might synergize with other tools for the correction or complementation of large or complex human pathogenic alleles.}, }
@article {pmid34719304, year = {2022}, author = {Zocca, VFB and Corrêa, GG and Lins, MRDCR and de Jesus, VN and Tavares, LF and Amorim, LADS and Kundlatsch, GE and Pedrolli, DB}, title = {The CRISPR toolbox for the gram-positive model bacterium Bacillus subtilis.}, journal = {Critical reviews in biotechnology}, volume = {42}, number = {6}, pages = {813-826}, doi = {10.1080/07388551.2021.1983516}, pmid = {34719304}, issn = {1549-7801}, mesh = {*Bacillus subtilis/genetics ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; }, abstract = {CRISPR has revolutionized the way we engineer genomes. Its simplicity and modularity have enabled the development of a great number of tools to edit genomes and to control gene expression. This powerful technology was first adapted to Bacillus subtilis in 2016 and has been intensely upgraded since then. Many tools have been successfully developed to build a CRISPR toolbox for this Gram-positive model and important industrial chassis. The toolbox includes tools, such as double-strand and single-strand cutting CRISPR for point mutation, gene insertion, and gene deletion up to 38 kb. Moreover, catalytic dead Cas proteins have been used for base editing, as well as for the control of gene expression (CRISPRi and CRISPRa). Many of these tools have been used for multiplex CRISPR with the most successful one targeting up to six loci simultaneously for point mutation. However, tools for efficient multiplex CRISPR for other functionalities are still missing in the toolbox. CRISPR engineering has already resulted in efficient protein and metabolite-producing strains, demonstrating its great potential. In this review, we cover all the important additions made to the B. subtilis CRISPR toolbox since 2016, and strain developments fomented by the technology.}, }
@article {pmid34349239, year = {2022}, author = {Gao, C and Wu, P and Yu, L and Liu, L and Liu, H and Tan, X and Wang, L and Huang, X and Wang, H}, title = {The application of CRISPR/Cas9 system in cervical carcinogenesis.}, journal = {Cancer gene therapy}, volume = {29}, number = {5}, pages = {466-474}, pmid = {34349239}, issn = {1476-5500}, support = {81830074//National Natural Science Foundation of China (National Science Foundation of China)/ ; 81902667//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {Animals ; CRISPR-Cas Systems ; Carcinogenesis/genetics ; Female ; Human papillomavirus 16/genetics/metabolism ; Humans ; Mice ; Mice, Transgenic ; *Oncogene Proteins, Viral/genetics ; Papillomavirus E7 Proteins/genetics ; *Papillomavirus Infections/complications/genetics/therapy ; *Precancerous Conditions/genetics ; Transcription Activator-Like Effector Nucleases/genetics/metabolism ; *Uterine Cervical Neoplasms/genetics/pathology/therapy ; }, abstract = {Integration of high-risk HPV genomes into cellular chromatin has been confirmed to promote cervical carcinogenesis, with HPV16 being the most prevalent high-risk type. Herein, we evaluated the therapeutic effect of the CRISPR/Cas9 system in cervical carcinogenesis, especially for cervical precancerous lesions. In cervical cancer/pre-cancer cell lines, we transfected the HPV16 E7 targeted CRISPR/Cas9, TALEN, ZFN plasmids, respectively. Compared to previous established ZFN and TALEN systems, CRISPR/Cas9 has shown comparable efficiency and specificity in inhibiting cell growth and colony formation and inducing apoptosis in cervical cancer/pre-cancer cell lines, which seemed to be more pronounced in the S12 cell line derived from the low-grade cervical lesion. Furthermore, in xenograft formation assays, CRISPR/Cas9 inhibited tumor formation of the S12 cell line in vivo and affected the corresponding protein expression. In the K14-HPV16 transgenic mice model of HPV-driven spontaneous cervical carcinogenesis, cervical application of CRISPR/Cas9 treatment caused mutations of the E7 gene and restored the expression of RB, E2F1, and CDK2, thereby reversing the cervical carcinogenesis phenotype. In this study, we have demonstrated that CRISPR/Cas9 targeting HPV16 E7 could effectively revert the HPV-related cervical carcinogenesis in vitro, as well as in K14-HPV16 transgenic mice, which has shown great potential in clinical treatment for cervical precancerous lesions.}, }
@article {pmid33864024, year = {2022}, author = {Marayati, R and Stafman, LL and Williams, AP and Bownes, LV and Quinn, CH and Markert, HR and Easlick, JL and Stewart, JE and Crossman, DK and Mroczek-Musulman, E and Beierle, EA}, title = {CRISPR/Cas9-mediated knockout of PIM3 suppresses tumorigenesis and cancer cell stemness in human hepatoblastoma cells.}, journal = {Cancer gene therapy}, volume = {29}, number = {5}, pages = {558-572}, pmid = {33864024}, issn = {1476-5500}, support = {T32 CA183926/CA/NCI NIH HHS/United States ; P30 CA013148/CA/NCI NIH HHS/United States ; CA013148//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; P30 AR048311/AR/NIAMS NIH HHS/United States ; T32 CA091078/CA/NCI NIH HHS/United States ; 5T32GM008361//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; T32 GM008361/GM/NIGMS NIH HHS/United States ; P30 AI027767/AI/NIAID NIH HHS/United States ; T32 CA229102/CA/NCI NIH HHS/United States ; T32 CA229102/CA/NCI NIH HHS/United States ; T32 CA091078/CA/NCI NIH HHS/United States ; T32 CA183926/CA/NCI NIH HHS/United States ; T32 CA229102/CA/NCI NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems ; Carcinogenesis/genetics ; Cell Line, Tumor ; Cell Proliferation/genetics ; Cell Transformation, Neoplastic/genetics ; Child ; *Hepatoblastoma/genetics/metabolism/pathology ; Humans ; *Liver Neoplasms/metabolism ; Mice ; Protein Serine-Threonine Kinases/genetics ; Proto-Oncogene Proteins/genetics ; }, abstract = {Hepatoblastoma remains one of the most difficult childhood tumors to treat and is alarmingly understudied. We previously demonstrated that Proviral Insertion site in Maloney murine leukemia virus (PIM) kinases, specifically PIM3, are overexpressed in human hepatoblastoma cells and function to promote tumorigenesis. We aimed to use CRISPR/Cas9 gene editing with dual gRNAs to introduce large inactivating deletions in the PIM3 gene and achieve stable PIM3 knockout in the human hepatoblastoma cell line, HuH6. PIM3 knockout of hepatoblastoma cells led to significantly decreased proliferation, viability, and motility, inhibited cell-cycle progression, decreased tumor growth in a xenograft murine model, and increased animal survival. Analysis of RNA sequencing data revealed that PIM3 knockout downregulated expression of pro-migratory and pro-invasive genes and upregulated expression of genes involved in apoptosis and differentiation. Furthermore, PIM3 knockout decreased hepatoblastoma cancer cell stemness as evidenced by decreased tumorsphere formation, decreased mRNA abundance of stemness markers, and decreased cell surface expression of CD133, a marker of hepatoblastoma stem cell-like cancer cells. Reintroduction of PIM3 into PIM3 knockout cells rescued the malignant phenotype. Successful CRISPR/Cas9 knockout of PIM3 kinase in human hepatoblastoma cells confirmed the role of PIM3 in promoting hepatoblastoma tumorigenesis and cancer cell stemness.}, }
@article {pmid35584409, year = {2022}, author = {Lee, HJ and Kim, HJ and Park, YJ and Lee, SJ}, title = {Efficient Single-Nucleotide Microbial Genome Editing Achieved Using CRISPR/Cpf1 with Maximally 3'-End-Truncated crRNAs.}, journal = {ACS synthetic biology}, volume = {}, number = {}, pages = {}, doi = {10.1021/acssynbio.2c00054}, pmid = {35584409}, issn = {2161-5063}, abstract = {Mismatch tolerance, a cause of the off-target effect, impedes accurate genome editing with the CRISPR/Cas system. Herein, we observed that oligonucleotide-directed single-base substitutions could be rarely introduced in the microbial genome using CRISPR/Cpf1-mediated negative selection. Because crRNAs have the ability to recognize and discriminate among specific target DNA sequences, we systematically compared the effects of modified crRNAs with 3'-end nucleotide truncations and a single mismatch on the genomic cleavage activity of FnCpf1 inEscherichia coli. Five nucleotides could be maximally truncated at the crRNA 3'-end for the efficient cleavage of the DNA targets of galK and xylB in the cells. However, target cleavage in the genome was inefficient when a single mismatch was simultaneously introduced in the maximally 3'-end-truncated crRNA. Based on these results, we assumed that the maximally truncated crRNA-Cpf1 complex can distinguish between single-base-edited and unedited targets in vivo. Compared to other crRNAs with shorter truncations, maximally 3'-end-truncated crRNAs showed highly efficient single-base substitutions (>80%) in the DNA targets of galK and xylB. Furthermore, the editing efficiency for the 24 bases in both galK and xylB showed success rates of 79 and 50%, respectively. We successfully introduced single-nucleotide indels in galK and xylB with editing efficiencies of 79 and 62%, respectively. Collectively, the maximally truncated crRNA-Cpf1 complex could perform efficient base and nucleotide editing regardless of the target base location or mutation type; this system is a simple and efficient tool for microbial genome editing, including indel correction, at the single-nucleotide resolution.}, }
@article {pmid35583743, year = {2022}, author = {Vento, JM and Beisel, CL}, title = {Genome Editing with Cas9 in Lactobacilli.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2479}, number = {}, pages = {245-261}, pmid = {35583743}, issn = {1940-6029}, abstract = {The bacterial genus Lactobacillus comprises a vast range of strains with varying metabolic and probiotic traits, with genome editing representing an essential tool to probe genotype-phenotype relationships and enhance their beneficial properties. Currently, one of the most effective means of genome editing in bacteria couples low-efficiency recombineering with high-efficiency counterselection by nucleases from CRISPR-Cas systems. In lactobacilli, several CRISPR-based genome editing methods exist that have shown varying success in different strains. Here, we detail a fast and simple approach using two shuttle vectors encoding a recombineering template as well as the Streptococcus pyogenes Cas9, a trans-activating RNA, and a CRISPR array. We provide a step-by-step procedure for cloning the shuttle vectors, sequentially transforming the vectors into lactobacilli, screening for the desired edit, and finally clearing the shuttle vectors from the mutant strain. As CRISPR-based genome editing in bacteria can fail for various reasons, we also lay out instructions for probing mechanisms of escape. Finally, we include practical notes along the way to facilitate each stage of genome editing, and we illustrate the technique using a representative edit in a strain of Lactobacillus plantarum. Overall, this method should serve as a complete guide to performing genome editing in lactobacilli.}, }
@article {pmid35583742, year = {2022}, author = {Xu, T and Tao, X and Kempher, ML and Zhou, J}, title = {Cas9 Nickase-Based Genome Editing in Clostridium cellulolyticum.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2479}, number = {}, pages = {227-243}, pmid = {35583742}, issn = {1940-6029}, abstract = {Clostridium cellulolyticum is a model mesophilic, cellulolytic bacterium, with the potential to produce biofuels from lignocellulose. However, the natural cellulose utilization efficiency is quite low and, therefore, metabolically engineered strains with increased efficiency can decrease both the overall cost and time required for biofuel production. Traditional genetic tools are inefficient, expensive, and time-consuming, but recent developments in the use of CRISPR-Cas genetic editing systems have greatly expanded our ability to reprogram cells. Here we describe an established protocol enabling one-step versatile genome editing in C. cellulolyticum. It integrates Cas9 nickase (Cas9n) which introduces a single nick that triggers repair via homologous recombination (SNHR) to edit genomic loci with high efficiency and accuracy. This one-step editing is achieved by transforming an all-in-one vector to coexpress Cas9n and a single guide RNA (gRNA) and carries a user-defined homologous donor template to promote SNHR at a desired target site. Additionally, this system has high specificity and allows for various types of genomic editing, including markerless insertions, deletions, substitutions, and even multiplex editing.}, }
@article {pmid35583741, year = {2022}, author = {Tan, LL and Heng, E and Zulkarnain, N and Hsiao, WC and Wong, FT and Zhang, MM}, title = {CRISPR/Cas-Mediated Genome Editing of Streptomyces.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2479}, number = {}, pages = {207-225}, pmid = {35583741}, issn = {1940-6029}, abstract = {Streptomyces are an important source and reservoir of natural products with diverse applications in medicine, agriculture, and food. Engineered Streptomyces strains have also proven to be functional chassis for the discovery and production of bioactive compounds and enzymes. However, genetic engineering of Streptomyces is often laborious and time-consuming. Here we describe protocols for CRISPR/Cas-mediated genome editing of Streptomyces. Starting from the design and assembly of all-in-one CRISPR/Cas constructs for efficient double-strand break-mediated genome editing, we also present protocols for intergeneric conjugation, CRISPR/Cas plasmid curing, and validation of edited strains.}, }
@article {pmid35583202, year = {2022}, author = {Luo, G and Najafi, J and Correia, PMP and Trinh, MDL and Chapman, EA and Østerberg, JT and Thomsen, HC and Pedas, PR and Larson, S and Gao, C and Poland, J and Knudsen, S and DeHaan, L and Palmgren, M}, title = {Accelerated Domestication of New Crops: Yield is Key.}, journal = {Plant &amp; cell physiology}, volume = {}, number = {}, pages = {}, doi = {10.1093/pcp/pcac065}, pmid = {35583202}, issn = {1471-9053}, support = {DEEPROOTS//Innovationsfonden/ ; LESSISMORE//Innovationsfonden/ ; CF18-1113//Carlsbergfondet/ ; 2019OC53580//Novo Nordisk Fonden/ ; }, abstract = {Sustainable agriculture in the future will depend on crops that are tolerant to biotic and abiotic stresses, require minimal input of water and nutrients, and can be cultivated with a minimal carbon footprint. Wild plants that fulfil these requirements abound in nature but are typically low yielding. Thus, replacing current high-yielding crops with less productive but resilient species will require the intractable trade-off of increasing land area under cultivation to produce the same yield. Cultivating more land reduces natural resources, reduces biodiversity, and increases our carbon footprint. Sustainable intensification can be achieved by increasing yield in underutilized or wild plant species that are already resilient but achieving this goal by conventional breeding programs may be a long-term prospect. De novo domestication of orphan or crop wild relatives using mutagenesis is an alternative and fast approach to achieve resilient crops with high yield. With new precise molecular techniques it should be possible to reach economically sustainable yields in a much shorter period of time than ever before in the history of agriculture.}, }
@article {pmid35581233, year = {2022}, author = {Sansbury, BM and Hewes, AM and Tharp, OM and Masciarelli, SB and Kaouser, S and Kmiec, EB}, title = {Homology directed correction, a new pathway model for point mutation repair catalyzed by CRISPR-Cas.}, journal = {Scientific reports}, volume = {12}, number = {1}, pages = {8132}, pmid = {35581233}, issn = {2045-2322}, abstract = {Gene correction is often referred to as the gold standard for precise gene editing and while CRISPR-Cas systems continue to expand the toolbox for clinically relevant genetic repair, mechanistic hurdles still hinder widespread implementation. One of the most prominent challenges to precise CRISPR-directed point mutation repair centers on the prevalence of on-site mutagenesis, wherein insertions and deletions appear at the targeted site following correction. Here, we introduce a pathway model for Homology Directed Correction, specifically point mutation repair, which enables a foundational analysis of genetic tools and factors influencing precise gene editing. To do this, we modified an in vitro gene editing system which utilizes a cell-free extract, CRISPR-Cas RNP and donor DNA template to catalyze point mutation repair. We successfully direct correction of four unique point mutations which include two unique nucleotide mutations at two separate targeted sites and visualize the repair profiles resulting from these reactions. This extension of the cell-free gene editing system to model point mutation repair may provide insight for understanding the factors influencing precise point mutation correction.}, }
@article {pmid35579071, year = {2022}, author = {Zhang, L and Zhao, X and Hu, X and Zhang, Y and Liu, R and Peng, H and Chen, Y and Zhang, H and Luo, Y}, title = {Probing low abundant DNA methylation by CRISPR-Cas12a-assisted cascade exponential amplification.}, journal = {The Analyst}, volume = {}, number = {}, pages = {}, doi = {10.1039/d2an00170e}, pmid = {35579071}, issn = {1364-5528}, abstract = {Aberrant DNA methylation plays a pivotal role in tumor development and metastasis, and is regarded as a valuable non-invasive cancer biomarker. However, the sensitive and accurate quantification of DNA methylation from clinical samples remains a challenge. Herein, we propose an easy-to-operate Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas system Assisted Methylation (CAM) approach for the sensitive detection of DNA methylation through the integration of rolling circle amplification and CRISPR-Cas12a-assisted cascade amplification. Briefly, bisulfite was employed to prepare the clinical samples so that the methylated DNA sequences trigger the subsequent triple signal amplifications, whilst the normal counterparts do not. The triple signal amplification procedure consists of methylated DNA sequence-based rolling circle amplification for a preliminary signal enhancement, a nicking enzyme-initiated target cleavage for a secondary amplification, and CRISPR-Cas12a enzyme-mediated trans-cleavage for a tertiary signal enhancement. This proposed approach reveals high sensitivity, which can even distinguish as low as 0.01% methylation levels from mixtures, paving the way towards the acceleration of methylation-based cancer diagnostics and management.}, }
@article {pmid35577099, year = {2022}, author = {Panda, G and Ray, A}, title = {Decrypting the mechanistic basis of CRISPR/Cas9 protein.}, journal = {Progress in biophysics and molecular biology}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.pbiomolbio.2022.05.001}, pmid = {35577099}, issn = {1873-1732}, abstract = {CRISPR/Cas system, a newly but extensively investigated genome-editing method, harbors practical solutions for various genetic problems. It relies on short guide RNAs (gRNAs) to recruit the Cas9 protein, a DNA cleaving enzyme, to its genomic target DNAs. The Cas9 enzyme exhibits some unique properties, like the ability to differentiate self vs. non-self - DNA strands using the base-pairing potential of crRNA, i.e., only CRISPR DNA is entirely complementary to the CRISPR repeat sequences at the crRNA whereas the presence of mismatches in the upstream region of the spacer permit CRISPR interference which is inhibited in case of CRISPR-DNA, allosteric regulation in its domains, and domain reorientation on sgRNA binding. Several groups have contributed their efforts in understanding the functioning of the CRISPR/Cas system, but even then, there is a lot more to explore in this area. The structural and sequence-based understanding of the whole CRISPR-associated bacterial ortholog family landscape is still ambiguous. A better understanding of the underlying energetics of the CRISPR/Cas9 system should reveal critical parameters to design better CRISPR/Cas9s.}, }
@article {pmid35573787, year = {2022}, author = {Shankar, C and Vasudevan, K and Jacob, JJ and Baker, S and Isaac, BJ and Neeravi, AR and Sethuvel, DPM and George, B and Veeraraghavan, B}, title = {Hybrid Plasmids Encoding Antimicrobial Resistance and Virulence Traits Among Hypervirulent Klebsiella pneumoniae ST2096 in India.}, journal = {Frontiers in cellular and infection microbiology}, volume = {12}, number = {}, pages = {875116}, doi = {10.3389/fcimb.2022.875116}, pmid = {35573787}, issn = {2235-2988}, mesh = {Anti-Bacterial Agents/pharmacology ; Drug Resistance, Bacterial ; Humans ; *Klebsiella Infections ; *Klebsiella pneumoniae ; Plasmids/genetics ; Virulence/genetics ; beta-Lactamases/genetics ; }, abstract = {Background: Hypervirulent variants of Klebsiella pneumoniae (HvKp) were typically associated with a broadly antimicrobial susceptible clone of sequence type (ST) 23 at the time of its emergence. Concerningly, HvKp is now also emerging within multidrug-resistant (MDR) clones, including ST11, ST15, and ST147. MDR-HvKp either carry both the virulence and resistance plasmids or carry a large hybrid plasmid coding for both virulence and resistance determinants. Here, we aimed to genetically characterize a collection of MDR-HvKp ST2096 isolates haboring hybrid plasmids carrying both antimicrobial resistance (AMR) and virulence genes.<br><br>Methods: Nine K. pneumoniae ST2096 isolated over 1 year from the blood sample of hospitalized patients in southern India that were MDR and suspected to be HvKp were selected. All nine isolates were subjected to short-read whole-genome sequencing; a subset (n = 4) was additionally subjected to long-read sequencing to obtain complete genomes for characterization. Mucoviscosity assay was also performed for phenotypic assessment.<br><br>Results: Among the nine isolates, seven were carbapenem-resistant, two of which carried bla NDM-5 on an IncFII plasmid and five carried bla OXA-232 on a ColKP3 plasmid. The organisms were confirmed as HvKp, with characteristic virulence genes (rmpA2, iutA, and iucABCD) carried on a large (~320 kbp) IncFIB-IncHI1B co-integrate. This hybrid plasmid also carried the aadA2, armA, bla OXA-1, msrE, mphE, sul1, and dfrA14 AMR genes in addition to the heavy-metal resistance genes. The hybrid plasmid showed about 60% similarity to the IncHI1B virulence plasmid of K. pneumoniae SGH10 and ~70% sequence identity with the first identified IncHI1B pNDM-MAR plasmid. Notably, the hybrid plasmid carried its type IV-A3 CRISPR-Cas system which harbored spacer regions against traL of IncF plasmids, thereby preventing their acquisition.<br><br>Conclusion: The convergence of virulence and AMR is clinically concerning in K. pneumoniae. Our data highlight the role of hybrid plasmids carrying both AMR and virulence genes in K. pneumoniae ST2096, suggesting that MDR-HvKp is not confined to selected clones; we highlight the continued emergence of such genotypes across the species. The convergence is occurring globally amidst several clones and is of great concern to public health.}, }
@article {pmid35573047, year = {2022}, author = {Kath, J and Du, W and Pruene, A and Braun, T and Thommandru, B and Turk, R and Sturgeon, ML and Kurgan, GL and Amini, L and Stein, M and Zittel, T and Martini, S and Ostendorf, L and Wilhelm, A and Akyüz, L and Rehm, A and Höpken, UE and Pruß, A and Künkele, A and Jacobi, AM and Volk, HD and Schmueck-Henneresse, M and Stripecke, R and Reinke, P and Wagner, DL}, title = {Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cells.}, journal = {Molecular therapy. Methods &amp; clinical development}, volume = {25}, number = {}, pages = {311-330}, doi = {10.1016/j.omtm.2022.03.018}, pmid = {35573047}, issn = {2329-0501}, abstract = {Chimeric antigen receptor (CAR) redirected T cells are potent therapeutic options against hematological malignancies. The current dominant manufacturing approach for CAR T cells depends on retroviral transduction. With the advent of gene editing, insertion of a CD19-CAR into the T cell receptor (TCR) alpha constant (TRAC) locus using adeno-associated viruses for gene transfer was demonstrated, and these CD19-CAR T cells showed improved functionality over their retrovirally transduced counterparts. However, clinical-grade production of viruses is complex and associated with extensive costs. Here, we optimized a virus-free genome-editing method for efficient CAR insertion into the TRAC locus of primary human T cells via nuclease-assisted homology-directed repair (HDR) using CRISPR-Cas and double-stranded template DNA (dsDNA). We evaluated DNA-sensor inhibition and HDR enhancement as two pharmacological interventions to improve cell viability and relative CAR knockin rates, respectively. While the toxicity of transfected dsDNA was not fully prevented, the combination of both interventions significantly increased CAR knockin rates and CAR T cell yield. Resulting TRAC-replaced CD19-CAR T cells showed antigen-specific cytotoxicity and cytokine production in vitro and slowed leukemia progression in a xenograft mouse model. Amplicon sequencing did not reveal significant indel formation at potential off-target sites with or without exposure to DNA-repair-modulating small molecules. With TRAC-integrated CAR+ T cell frequencies exceeding 50%, this study opens new perspectives to exploit pharmacological interventions to improve non-viral gene editing in T cells.}, }
@article {pmid35572739, year = {2022}, author = {Abdullah, M and Okemo, P and Furtado, A and Henry, R}, title = {Potential of Genome Editing to Capture Diversity From Australian Wild Rice Relatives.}, journal = {Frontiers in genome editing}, volume = {4}, number = {}, pages = {875243}, doi = {10.3389/fgeed.2022.875243}, pmid = {35572739}, issn = {2673-3439}, abstract = {Rice, a staple food worldwide and a model crop, could benefit from the introduction of novel genetics from wild relatives. Wild rice in the AA genome group closely related to domesticated rice is found across the tropical world. Due to their locality outside the range of domesticated rice, Australian wild rice populations are a potential source of unique traits for rice breeding. These rice species provide a diverse gene pool for improvement that could be utilized for desirable traits such as stress resistance, disease tolerance, and nutritional qualities. However, they remain poorly characterized. The CRISPR/Cas system has revolutionized gene editing and has improved our understanding of gene functions. Coupled with the increasing availability of genomic information on the species, genes in Australian wild rice could be modified through genome editing technologies to produce new domesticates. Alternatively, beneficial alleles from these rice species could be incorporated into cultivated rice to improve critical traits. Here, we summarize the beneficial traits in Australian wild rice, the available genomic information and the potential of gene editing to discover and understand the functions of novel alleles. Moreover, we discuss the potential domestication of these wild rice species for health and economic benefits to rice production globally.}, }
@article {pmid35572197, year = {2022}, author = {Nieland, L and van Solinge, TS and Cheah, PS and Morsett, LM and El Khoury, J and Rissman, JI and Kleinstiver, BP and Broekman, MLD and Breakefield, XO and Abels, ER}, title = {CRISPR-Cas knockout of miR21 reduces glioma growth.}, journal = {Molecular therapy oncolytics}, volume = {25}, number = {}, pages = {121-136}, doi = {10.1016/j.omto.2022.04.001}, pmid = {35572197}, issn = {2372-7705}, abstract = {Non-coding RNAs, including microRNAs (miRNAs), support the progression of glioma. miR-21 is a small, non-coding transcript involved in regulating gene expression in multiple cellular pathways, including the regulation of proliferation. High expression of miR-21 has been shown to be a major driver of glioma growth. Manipulating the expression of miRNAs is a novel strategy in the development of therapeutics in cancer. In this study we aimed to target miR-21. Using CRISPR genome-editing technology, we disrupted the miR-21 coding sequences in glioma cells. Depletion of this miRNA resulted in the upregulation of many downstream miR-21 target mRNAs involved in proliferation. Phenotypically, CRISPR-edited glioma cells showed reduced migration, invasion, and proliferation in vitro. In immunocompetent mouse models, miR-21 knockout tumors showed reduced growth resulting in an increased overall survival. In summary, we show that by knocking out a key miRNA in glioma, these cells have decreased proliferation capacity both in vitro and in vivo. Overall, we identified miR-21 as a potential target for CRISPR-based therapeutics in glioma.}, }
@article {pmid35572153, year = {2022}, author = {Christian, A}, title = {Addressing Conflicts of Interest and Conflicts of Commitment in Public Advocacy and Policy Making on CRISPR/Cas-Based Human Genome Editing.}, journal = {Frontiers in research metrics and analytics}, volume = {7}, number = {}, pages = {775336}, doi = {10.3389/frma.2022.775336}, pmid = {35572153}, issn = {2504-0537}, abstract = {Leading experts on CRISPR/Cas-based genome editing-such as 2020 Nobel laureates Jennifer Doudna and Emmanuelle Charpentier-are not only renowned specialists in their fields, but also public advocates for upcoming regulatory frameworks on CRISPR/Cas. These frameworks will affect large portions of biomedical research on human genome editing. In advocating for particular ways of handling the risks and prospects of this technology, high-profile scientists not only serve as scientific experts, but also as moral advisers. The majority of them currently intend to bring about a "responsible pathway" toward human genome interventions in clinical therapy. Engaging in advocacy for such a pathway, they issue moral judgments on the risks and benefits of this new technology. They declare that there actually is a responsible pathway, they draft resolutions on temporary moratoria, they make judgments on which groups and individuals are credible and should participate in public and semi-public debates, so they also set the standards for deciding who counts as well-informed, as well as the standards of evidence for adopting or rejecting research policies. This degree of influence on public debates and policy making is, at the very least, noteworthy. This contribution sounds a note of caution with regard to the endeavor of a responsible pathway to human genome editing and in particular scrutinizes the legitimacy of expert-driven research policies given commercial conflicts of interest and conflicts of commitment among first-rank scholars.}, }
@article {pmid35569864, year = {2022}, author = {Islam, MM and Koirala, D}, title = {Toward a next-generation diagnostic tool: A review on emerging isothermal nucleic acid amplification techniques for the detection of SARS-CoV-2 and other infectious viruses.}, journal = {Analytica chimica acta}, volume = {1209}, number = {}, pages = {339338}, doi = {10.1016/j.aca.2021.339338}, pmid = {35569864}, issn = {1873-4324}, abstract = {As the COVID-19 pandemic continues to affect human health across the globe rapid, simple, point-of-care (POC) diagnosis of infectious viruses such as SARS-CoV-2 remains challenging. Polymerase chain reaction (PCR)-based diagnosis has risen to meet these demands and despite its high-throughput and accuracy, it has failed to gain traction in the rapid, low-cost, point-of-test settings. In contrast, different emerging isothermal amplification-based detection methods show promise in the rapid point-of-test market. In this comprehensive study of the literature, several promising isothermal amplification methods for the detection of SARS-CoV-2 are critically reviewed that can also be applied to other infectious viruses detection. Starting with a brief discussion on the SARS-CoV-2 structure, its genomic features, and the epidemiology of the current pandemic, this review focuses on different emerging isothermal methods and their advancement. The potential of isothermal amplification combined with the revolutionary CRISPR/Cas system for a more powerful detection tool is also critically reviewed. Additionally, the commercial success of several isothermal methods in the pandemic are highlighted. Different variants of SARS-CoV-2 and their implication on isothermal amplifications are also discussed. Furthermore, three most crucial aspects in achieving a simple, fast, and multiplexable platform are addressed.}, }
@article {pmid35568950, year = {2022}, author = {Cai, P and Han, M and Zhang, R and Ding, S and Zhang, D and Liu, D and Liu, S and Hu, QN}, title = {SynBioStrainFinder: A microbial strain database of manually curated CRISPR/Cas genetic manipulation system information for biomanufacturing.}, journal = {Microbial cell factories}, volume = {21}, number = {1}, pages = {87}, pmid = {35568950}, issn = {1475-2859}, support = {2019YFA0904300//National Key Research and Development Program of China/ ; 31700081//National Natural Science Foundation of China/ ; 31570092//National Natural Science Foundation of China/ ; QYZDB-SSW-SMC012//CAS STS program/ ; 153D31KYSB20170121//International Partnership Program of Chinese Academy of Sciences of China/ ; }, mesh = {*CRISPR-Cas Systems ; *Gene Editing ; }, abstract = {BACKGROUND: Microbial strain information databases provide valuable data for microbial basic research and applications. However, they rarely contain information on the genetic operating system of microbial strains.<br><br>RESULTS: We established a comprehensive microbial strain database, SynBioStrainFinder, by integrating CRISPR/Cas gene-editing system information with cultivation methods, genome sequence data, and compound-related information. It is presented through three modules, Strain2Gms/PredStrain2Gms, Strain2BasicInfo, and Strain2Compd, which combine to form a rapid strain information query system conveniently curated, integrated, and accessible on a single platform. To date, 1426 CRISPR/Cas gene-editing records of 157 microbial strains have been manually extracted from the literature in the Strain2Gms module. For strains without established CRISPR/Cas systems, the PredStrain2Gms module recommends the system of the most closely related strain as a reference to facilitate the construction of a new CRISPR/Cas gene-editing system. The database contains 139,499 records of strain cultivation and genome sequences, and 773,298 records of strain-related compounds. To facilitate simple and intuitive data application, all microbial strains are also labeled with stars based on the order and availability of strain information. SynBioStrainFinder provides a user-friendly interface for querying, browsing, and visualizing detailed information on microbial strains, and it is publicly available at http://design.rxnfinder.org/biosynstrain/ .<br><br>CONCLUSION: SynBioStrainFinder is the first microbial strain database with manually curated information on the strain CRISPR/Cas system as well as other microbial strain information. It also provides reference information for the construction of new CRISPR/Cas systems. SynBioStrainFinder will serve as a useful resource to extend microbial strain research and application for biomanufacturing.}, }
@article {pmid35568789, year = {2022}, author = {Chaudhary, M and Mukherjee, TK and Singh, R and Gupta, M and Goyal, S and Singhal, P and Kumar, R and Bhusal, N and Sharma, P}, title = {CRISPR/Cas technology for improving nutritional values in the agricultural sector: an update.}, journal = {Molecular biology reports}, volume = {}, number = {}, pages = {}, pmid = {35568789}, issn = {1573-4978}, abstract = {BACKGROUND: The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system was initially identified in bacteria and archaea as a defense mechanism to confer immunity against phages. Later on, it was developed as a gene editing tool for both prokaryotic and eukaryotic cells including plant cells.<br><br>METHODS AND RESULTS: CRISPR/Cas9 approach has wider applications in reverse genetics as well as in crop improvement. Various characters involved in enhancing economic value and crop sustainability against biotic/abiotic stresses can be targeted through this tool. Currently, CRISPR/Cas9 gene editing mechanism has been applied on around 20 crop species for improvement in several traits including yield enhancement and resistance against biotic and abiotic stresses. In the last five years, maximum genome editing research has been validated in rice, wheat, maize and soybean. Genes targeted in these plants has been involved in causing male sterility, conferring resistance against pathogens or having certain nutritional value.<br><br>CONCLUSIONS: Current review summarizes various applications of CRISPR/Cas system and its future prospects in plant biotechnology targeting crop improvement with higher yield, disease tolerance and enhanced nutritional value.}, }
@article {pmid35565854, year = {2022}, author = {Quéré, M and Alberto, JM and Broly, F and Hergalant, S and Christov, C and Gauchotte, G and Guéant, JL and Namour, F and Battaglia-Hsu, SF}, title = {ALDH1L2 Knockout in U251 Glioblastoma Cells Reduces Tumor Sphere Formation by Increasing Oxidative Stress and Suppressing Methionine Dependency.}, journal = {Nutrients}, volume = {14}, number = {9}, pages = {}, pmid = {35565854}, issn = {2072-6643}, support = {NEGERE 2019//La Ligue Contre Cancer/ ; BMS Incitatif 2020//Université de Lorraine/ ; }, mesh = {Cell Line, Tumor ; *Glioblastoma/metabolism ; Humans ; Methionine/metabolism ; Neoplastic Stem Cells/metabolism ; Oxidative Stress ; Reactive Oxygen Species/metabolism ; }, abstract = {Previously, the in vitro growth of cancer stem cells in the form of tumor spheres from five different brain cancer cell lines was found to be methionine-dependent. As this earlier work indicated that ALDH1L2, a folate-dependent mitochondria aldehyde dehydrogenase gene, is upregulated in glioblastoma stem cells, we invalidated this gene using CRISPR-cas 9 technique in this present work. We reported here that this invalidation was effective in U251 glioblastoma cells, and no cas9 off target site could be detected by genome sequencing of the two independent knockout targeting either exon I or exon III. The knockout of ALDH1L2 gene in U251 cells rendered the growth of the cancer stem cells of U251 methionine independent. In addition, a much higher ROS (reactive oxygen radicals) level can be detected in the knockout cells compared to the wild type cells. Our evidence here linked the excessive ROS level of the knockout cells to reduced total cellular NADPH. Our evidence suggested also that the cause of the slower growth of the knockout turmor sphere may be related to its partial differentiation.}, }
@article {pmid35563876, year = {2022}, author = {Gómez-García, F and Martínez-Pulleiro, R and Carrera, N and Allegue, C and Garcia-Gonzalez, MA}, title = {Genetic Kidney Diseases (GKDs) Modeling Using Genome Editing Technologies.}, journal = {Cells}, volume = {11}, number = {9}, pages = {}, pmid = {35563876}, issn = {2073-4409}, support = {PI18/00378//Instituto de Salud Carlos III/ ; IN607B-2016/020//Axencia Galega de Innovacion/ ; ED431G 2019/02//Xunta de Galicia/ ; RD21/0005/0020//Redes de Investigación Cooperativa Orientadas a Resultados en Salud/ ; RD16/0009/0024//Red de Investigación Renal/ ; }, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Endonucleases/genetics ; Female ; *Gene Editing/methods ; Humans ; *Kidney Diseases/genetics/therapy ; Male ; Zinc Finger Nucleases ; }, abstract = {Genetic kidney diseases (GKDs) are a group of rare diseases, affecting approximately about 60 to 80 per 100,000 individuals, for which there is currently no treatment that can cure them (in many cases). GKDs usually leads to early-onset chronic kidney disease, which results in patients having to undergo dialysis or kidney transplant. Here, we briefly describe genetic causes and phenotypic effects of six GKDs representative of different ranges of prevalence and renal involvement (ciliopathy, glomerulopathy, and tubulopathy). One of the shared characteristics of GKDs is that most of them are monogenic. This characteristic makes it possible to use site-specific nuclease systems to edit the genes that cause GKDs and generate in vitro and in vivo models that reflect the genetic abnormalities of GKDs. We describe and compare these site-specific nuclease systems (zinc finger nucleases (ZFNs), transcription activator-like effect nucleases (TALENs) and regularly clustered short palindromic repeat-associated protein (CRISPR-Cas9)) and review how these systems have allowed the generation of cellular and animal GKDs models and how they have contributed to shed light on many still unknown fields in GKDs. We also indicate the main obstacles limiting the application of these systems in a more efficient way. The information provided here will be useful to gain an accurate understanding of the technological advances in the field of genome editing for GKDs, as well as to serve as a guide for the selection of both the genome editing tool and the gene delivery method most suitable for the successful development of GKDs models.}, }
@article {pmid35563479, year = {2022}, author = {Feser, CJ and Lees, CJ and Lammers, DT and Riddle, MJ and Bingham, JR and Eckert, MJ and Tolar, J and Osborn, MJ}, title = {Engineering CRISPR/Cas9 for Multiplexed Recombinant Coagulation Factor Production.}, journal = {International journal of molecular sciences}, volume = {23}, number = {9}, pages = {}, pmid = {35563479}, issn = {1422-0067}, support = {W81XWH-20-C-0052//US Special Operations Command/ ; }, mesh = {*Blood Coagulation Factors/genetics ; *CRISPR-Cas Systems ; Fibrinogen/genetics ; Gene Editing/methods ; HEK293 Cells ; Humans ; Transcriptional Activation ; }, abstract = {Current hemostatic agents are obtained from pooled plasma from multiple donors requiring costly pathogen screening and processing. Recombinant DNA-based production represents an engineering solution that could improve supply, uniformity, and safety. Current approaches are typically for single gene candidate peptides and often employ non-human cells. We devised an approach where multiple gene products could be produced from a single population of cells. We identified gene specific Synergistic Activation Mediators (SAM) from the CRISPR/Cas9 system for targeted overexpression of coagulation factors II, VII, IX, X, and fibrinogen. The components of the CRISPR-SAM system were expressed in Human Embryonic Kidney Cells (HEK293), and single (singleplex) or multi-gene (multiplex) upregulation was assessed by quantitative RT-PCR (qRT-PCR) and protein expression by ELISA analysis. Factor II, VII, IX, and X singleplex and multiplex activation resulted in 120-4700-fold and 60-680-fold increases in gene expression, respectively. Fibrinogen sub-unit gene activation resulted in a 1700-92,000-fold increases and 80-5500-fold increases in singleplex or multiplex approaches, respectively. ELISA analysis showed a concomitant upregulation of candidate gene products. Our findings demonstrate the capability of CRISPR/Cas9 SAMs for single or multi-agent production in human cells and represent an engineering advance that augments current recombinant peptide production techniques.}, }
@article {pmid35563453, year = {2022}, author = {Shin, NR and Shin, YH and Kim, HS and Park, YD}, title = {Function Analysis of the PR55/B Gene Related to Self-Incompatibility in Chinese Cabbage Using CRISPR/Cas9.}, journal = {International journal of molecular sciences}, volume = {23}, number = {9}, pages = {}, pmid = {35563453}, issn = {1422-0067}, support = {NRF-2021M3E5E6025387//National Research Foundation (NRF) of Korea funded by the Ministry of Science, ICT/ ; }, mesh = {*Brassica/genetics ; *CRISPR-Cas Systems ; China ; Gene Editing ; Mutagenesis ; Plant Breeding ; }, abstract = {Chinese cabbage, a major crop in Korea, shows self-incompatibility (SI). SI is controlled by the type 2A serine/threonine protein phosphatases (PP2As). The PP2A gene is controlled by regulatory subunits that comprise a 36 kDa catalyst C subunit, a 65 kDa regulatory A subunit, and a variety of regulatory B subunits (50-70 kDa). Among them, the PP2A 55 kDa B regulatory subunit (PR55/B) gene located in the A05 chromosome has 13 exons spanning 2.9 kb, and two homologous genes, Bra018924 and Bra014296, were found to be present on the A06 and A08 chromosome, respectively. In this study, we performed a functional analysis of the PR55/B gene using clustered regularly interspaced short palindromic repeats/CRISPR-associated system 9 (CRISPR/Cas9)-mediated gene mutagenesis. CRISPR/Cas9 technology can be used to easily introduce mutations in the target gene. Tentative gene-edited lines were generated by the Agrobacterium-mediated transfer and were selected by PCR and Southern hybridization analysis. Furthermore, pods were confirmed to be formed in flower pollination (FP) as well as bud pollination (BP) in some gene-edited lines. Seed fertility of gene-edited lines indicated that the PR55/B gene plays a key role in SI. Finally, self-compatible T-DNA-free T2 gene-edited plants and edited sequences of target genes were secured. The self-compatible Chinese cabbage developed in this study is expected to contribute to Chinese cabbage breeding.}, }
@article {pmid35563435, year = {2022}, author = {Fischer, B and Schmidt, V and Ly, TD and Kleine, A and Knabbe, C and Faust-Hinse, I}, title = {First Characterization of Human Dermal Fibroblasts Showing a Decreased Xylosyltransferase-I Expression Induced by the CRISPR/Cas9 System.}, journal = {International journal of molecular sciences}, volume = {23}, number = {9}, pages = {}, pmid = {35563435}, issn = {1422-0067}, support = {FA 1381/1-1//Deutsche Forschungsgemeinschaft/ ; }, mesh = {*CRISPR-Cas Systems ; *Fibroblasts/metabolism ; Gene Editing ; Humans ; Infant, Newborn ; Pentosyltransferases/genetics/metabolism ; Skin/metabolism ; }, abstract = {BACKGROUND: Xylosyltransferases-I and II (XT-I and XT-II) catalyze the initial and rate limiting step of the proteoglycan (PG) biosynthesis and therefore have an import impact on the homeostasis of the extracellular matrix (ECM). The reason for the occurrence of two XT-isoforms in all higher organisms remains unknown and targeted genome-editing strategies could shed light on this issue.<br><br>METHODS: XT-I deficient neonatal normal human dermal fibroblasts were generated by using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated proteins (Cas) 9 system. We analyzed if a reduced XT-I activity leads to abnormalities regarding ECM-composition, myofibroblast differentiation, cellular senescence and skeletal and cartilage tissue homeostasis.<br><br>RESULTS: We successfully introduced compound heterozygous deletions within exon 9 of the XYLT1 gene. Beside XYLT1, we detected altered gene-expression levels of further, inter alia ECM-related, genes. Our data further reveal a dramatically reduced XT-I protein activity. Abnormal myofibroblast-differentiation was demonstrated by elevated alpha-smooth muscle actin expression on both, mRNA- and protein level. In addition, wound-healing capability was slightly delayed. Furthermore, we observed an increased cellular-senescence of knockout cells and an altered expression of target genes knowing to be involved in skeletonization.<br><br>CONCLUSION: Our data show the tremendous relevance of the XT-I isoform concerning myofibroblast-differentiation and ECM-homeostasis as well as the pathophysiology of skeletal disorders.}, }
@article {pmid35563412, year = {2022}, author = {Yao, M and Ren, T and Pan, Y and Xue, X and Li, R and Zhang, L and Li, Y and Huang, K}, title = {A New Generation of Lineage Tracing Dynamically Records Cell Fate Choices.}, journal = {International journal of molecular sciences}, volume = {23}, number = {9}, pages = {}, pmid = {35563412}, issn = {1422-0067}, support = {32070654//National Natural Science Foundation of China/ ; 201901D211193//Shanxi Province Science Foundation for Outstanding Youths, China/ ; 32071454//National Natural Science Foundation of China/ ; }, mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Lineage/genetics ; Gene Editing ; *Single-Cell Analysis ; *Transcriptome ; }, abstract = {Reconstructing the development of lineage relationships and cell fate mapping has been a fundamental problem in biology. Using advanced molecular biology and single-cell RNA sequencing, we have profiled transcriptomes at the single-cell level and mapped cell fates during development. Recently, CRISPR/Cas9 barcode editing for large-scale lineage tracing has been used to reconstruct the pseudotime trajectory of cells and improve lineage tracing accuracy. This review presents the progress of the latest CbLT (CRISPR-based Lineage Tracing) and discusses the current limitations and potential technical pitfalls in their application and other emerging concepts.}, }
@article {pmid35563297, year = {2022}, author = {Moniruzzaman, M and Zhong, Y and Huang, Z and Zhong, G}, title = {Having a Same Type IIS Enzyme's Restriction Site on Guide RNA Sequence Does Not Affect Golden Gate (GG) Cloning and Subsequent CRISPR/Cas Mutagenesis.}, journal = {International journal of molecular sciences}, volume = {23}, number = {9}, pages = {}, pmid = {35563297}, issn = {1422-0067}, support = {2019B030316005//Guangdong Provincial Science and Technology Program/ ; 2018B020202009//Guangdong Provincial Science and Technology Program/ ; 32072535//National Natural Science Foundation of China/ ; 32002016//National Natural Science Foundation of China/ ; BZ201902//Dean's Foundation of Guangdong Academy of Agricultural Sciences/ ; 2020KJ108//Guangdong Provincial Special Fund for Modern Agriculture Industry Technology Innovation Special Teams/ ; }, mesh = {Base Sequence ; *CRISPR-Cas Systems/genetics ; Cloning, Molecular ; Gene Editing ; Mutagenesis ; *RNA, Guide/genetics ; }, abstract = {Golden gate/modular cloning facilitates faster and more efficient cloning by utilizing the unique features of the type IIS restriction enzymes. However, it is known that targeted insertion of DNA fragment(s) must not include internal type IIS restriction recognition sites. In the case of cloning CRISPR constructs by using golden gate (GG) cloning, this narrows down the scope of guide RNA (gRNA) picks because the selection of a good gRNA for successful genome editing requires some obligation of fulfillment, and it is unwanted if a good gRNA candidate cannot be picked only because it has an internal type IIS restriction recognition site. In this article, we have shown that the presence of a type IIS restriction recognition site in a gRNA does not affect cloning and subsequent genome editing. After each step of GG reactions, correct insertions of gRNAs were verified by colony color and restriction digestion and were further confirmed by sequencing. Finally, the final vector containing a Cas12a nuclease and four gRNAs was used for Agrobacterium-mediated citrus cell transformation. Sequencing of PCR amplicons flanking gRNA-2 showed a substitution (C to T) mutation in transgenic plants. The knowledge derived from this study could widen the scope of GG cloning, particularly of gRNAs selection for GG-mediated cloning into CRISPR vectors.}, }
@article {pmid35563243, year = {2022}, author = {Tian, J and Xing, B and Li, M and Xu, C and Huo, YX and Guo, S}, title = {Efficient Large-Scale and Scarless Genome Engineering Enables the Construction and Screening of Bacillus subtilis Biofuel Overproducers.}, journal = {International journal of molecular sciences}, volume = {23}, number = {9}, pages = {}, pmid = {35563243}, issn = {1422-0067}, support = {2019YFA0904104//National Key R&amp;D Program of China/ ; 2021YFC2100500//National Key R&amp;D Program of China/ ; }, mesh = {*Bacillus subtilis/genetics ; Biofuels ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genome, Bacterial ; Metabolic Engineering ; }, abstract = {Bacillus subtilis is a versatile microbial cell factory that can produce valuable proteins and value-added chemicals. Long fragment editing techniques are of great importance for accelerating bacterial genome engineering to obtain desirable and genetically stable host strains. Herein, we develop an efficient CRISPR-Cas9 method for large-scale and scarless genome engineering in the Bacillus subtilis genome, which can delete up to 134.3 kb DNA fragments, 3.5 times as long as the previous report, with a positivity rate of 100%. The effects of using a heterologous NHEJ system, linear donor DNA, and various donor DNA length on the engineering efficiencies were also investigated. The CRISPR-Cas9 method was then utilized for Bacillus subtilis genome simplification and construction of a series of individual and cumulative deletion mutants, which are further screened for overproducer of isobutanol, a new generation biofuel. These results suggest that the method is a powerful genome engineering tool for constructing and screening engineered host strains with enhanced capabilities, highlighting the potential for synthetic biology and metabolic engineering.}, }
@article {pmid35563030, year = {2022}, author = {Toinga-Villafuerte, S and Vales, MI and Awika, JM and Rathore, KS}, title = {CRISPR/Cas9-Mediated Mutagenesis of the Granule-Bound Starch Synthase Gene in the Potato Variety Yukon Gold to Obtain Amylose-Free Starch in Tubers.}, journal = {International journal of molecular sciences}, volume = {23}, number = {9}, pages = {}, pmid = {35563030}, issn = {1422-0067}, mesh = {Amylopectin/metabolism ; Amylose/metabolism ; CRISPR-Cas Systems/genetics ; Gold/metabolism ; Mutagenesis ; *Solanum tuberosum/genetics/metabolism ; Starch/metabolism ; *Starch Synthase/genetics ; Yukon Territory ; }, abstract = {Potato (Solanum tuberosum L.) is the third most important food crop after rice and wheat. Its tubers are a rich source of dietary carbohydrates in the form of starch, which has many industrial applications. Starch is composed of two polysaccharides, amylose and amylopectin, and their ratios determine different properties and functionalities. Potato varieties with higher amylopectin have many food processing and industrial applications. Using Agrobacterium-mediated transformation, we delivered Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) reagents to potato (variety Yukon Gold) cells to disrupt the granule-bound starch synthase (gbssI) gene with the aim of eliminating the amylose component of starch. Lugol-Iodine staining of the tubers showed a reduction or complete elimination of amylose in some of the edited events. These results were further confirmed by the perchloric acid and enzymatic methods. One event (T2-7) showed mutations in all four gbss alleles and total elimination of amylose from the tubers. Viscosity profiles of the tuber starch from six different knockout events were determined using a Rapid Visco Analyzer (RVA), and the values reflected the amylopectin/amylose ratio. Follow-up studies will focus on eliminating the CRISPR components from the events and on evaluating the potential of clones with various amylose/amylopectin ratios for food processing and other industrial applications.}, }
@article {pmid35562887, year = {2022}, author = {Wang, YY and Hsu, SH and Tsai, HY and Cheng, FY and Cheng, MC}, title = {Transcriptomic and Proteomic Analysis of CRISPR/Cas9-Mediated ARC-Knockout HEK293 Cells.}, journal = {International journal of molecular sciences}, volume = {23}, number = {9}, pages = {}, pmid = {35562887}, issn = {1422-0067}, mesh = {CRISPR-Cas Systems ; Carrier Proteins ; Chromatography, Liquid ; HEK293 Cells ; Humans ; Microfilament Proteins ; Mitochondrial Proteins ; *Proteomics ; Tandem Mass Spectrometry ; *Transcriptome ; }, abstract = {Arc/Arg3.1 (activity-regulated cytoskeletal-associated protein (ARC)) is a critical regulator of long-term synaptic plasticity and is involved in the pathophysiology of schizophrenia. The functions and mechanisms of human ARC action are poorly understood and worthy of further investigation. To investigate the function of the ARC gene in vitro, we generated an ARC-knockout (KO) HEK293 cell line via CRISPR/Cas9-mediated gene editing and conducted RNA sequencing and label-free LC-MS/MS analysis to identify the differentially expressed genes and proteins in isogenic ARC-KO HEK293 cells. Furthermore, we used bioluminescence resonance energy transfer (BRET) assays to detect interactions between the ARC protein and differentially expressed proteins. Genetic deletion of ARC disturbed multiple genes involved in the extracellular matrix and synaptic membrane. Seven proteins (HSPA1A, ENO1, VCP, HMGCS1, ALDH1B1, FSCN1, and HINT2) were found to be differentially expressed between ARC-KO cells and ARC wild-type cells. BRET assay results showed that ARC interacted with PSD95 and HSPA1A. Overall, we found that ARC regulates the differential expression of genes involved in the extracellular matrix, synaptic membrane, and heat shock protein family. The transcriptomic and proteomic profiles of ARC-KO HEK293 cells presented here provide new evidence for the mechanisms underlying the effects of ARC and molecular pathways involved in schizophrenia pathophysiology.}, }
@article {pmid35562427, year = {2022}, author = {Wang, JY and Pausch, P and Doudna, JA}, title = {Structural biology of CRISPR-Cas immunity and genome editing enzymes.}, journal = {Nature reviews. Microbiology}, volume = {}, number = {}, pages = {}, pmid = {35562427}, issn = {1740-1534}, abstract = {CRISPR-Cas systems provide resistance against foreign mobile genetic elements and have a wide range of genome editing and biotechnological applications. In this Review, we examine recent advances in understanding the molecular structures and mechanisms of enzymes comprising bacterial RNA-guided CRISPR-Cas immune systems and deployed for wide-ranging genome editing applications. We explore the adaptive and interference aspects of CRISPR-Cas function as well as open questions about the molecular mechanisms responsible for genome targeting. These structural insights reflect close evolutionary links between CRISPR-Cas systems and mobile genetic elements, including the origins and evolution of CRISPR-Cas systems from DNA transposons, retrotransposons and toxin-antitoxin modules. We discuss how the evolution and structural diversity of CRISPR-Cas systems explain their functional complexity and utility as genome editing tools.}, }
@article {pmid35560111, year = {2022}, author = {Worthington, AK and Forsberg, EC}, title = {A CRISPR View of Hematopoietic Stem Cells: Moving Innovative Bioengineering into the Clinic.}, journal = {American journal of hematology}, volume = {}, number = {}, pages = {}, doi = {10.1002/ajh.26588}, pmid = {35560111}, issn = {1096-8652}, abstract = {CRISPR/Cas genome engineering has emerged as a powerful tool to modify precise genomic sequences with unparalleled accuracy and efficiency. Major advances in CRISPR technologies over the last five years have fueled the development of novel techniques in hematopoiesis research to interrogate the complexities of hematopoietic stem cell (HSC) biology. In particular, high throughput CRISPR based screens using various "flavors" of Cas coupled with sequencing and/or functional outputs are becoming increasingly efficient and accessible. In this review, we discuss recent achievements in CRISPR-mediated genomic engineering and how these new tools have advanced the understanding of HSC heterogeneity and function throughout life. Additionally, we highlight how these techniques can be used to answer previously inaccessible questions and the challenges to implement them. Finally, we focus on their translational potential to both model and treat hematological diseases in the clinic. This article is protected by copyright. All rights reserved.}, }
@article {pmid35559022, year = {2022}, author = {Mandal, S and Ghorai, M and Anand, U and Roy, D and Kant, N and Mishra, T and Mane, AB and Jha, NK and Lal, MK and Tiwari, RK and Kumar, M and Radha,  and Ghosh, A and Bhattacharjee, R and Proćków, J and Dey, A}, title = {Cytokinins: A Genetic Target for Increasing Yield Potential in the CRISPR Era.}, journal = {Frontiers in genetics}, volume = {13}, number = {}, pages = {883930}, pmid = {35559022}, issn = {1664-8021}, abstract = {Over the last decade, remarkable progress has been made in our understanding the phytohormones, cytokinin's (CKs) biosynthesis, perception, and signalling pathways. Additionally, it became apparent that interfering with any of these steps has a significant effect on all stages of plant growth and development. As a result of their complex regulatory and cross-talk interactions with other hormones and signalling networks, they influence and control a wide range of biological activities, from cellular to organismal levels. In agriculture, CKs are extensively used for yield improvement and management because of their wide-ranging effects on plant growth, development and physiology. One of the primary targets in this regard is cytokinin oxidase/dehydrogenase (CKO/CKX), which is encoded by CKX gene, which catalyses the irreversible degradation of cytokinin. The previous studies on various agronomically important crops indicated that plant breeders have targeted CKX directly. In recent years, prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been increasingly used in editing the CKO/CKX gene and phenomenal results have been achieved. This review provides an updated information on the applications of CRISPR-based gene-editing tools in manipulating cytokinin metabolism at the genetic level for yield improvement. Furthermore, we summarized the current developments of RNP-mediated DNA/transgene-free genomic editing of plants which would broaden the application of this technology. The current review will advance our understanding of cytokinins and their role in sustainably increase crop production through CRISPR/Cas genome editing tool.}, }
@article {pmid35558825, year = {2022}, author = {Rahman, F and Mishra, A and Gupta, A and Sharma, R}, title = {Spatiotemporal Regulation of CRISPR/Cas9 Enables Efficient, Precise, and Heritable Edits in Plant Genomes.}, journal = {Frontiers in genome editing}, volume = {4}, number = {}, pages = {870108}, pmid = {35558825}, issn = {2673-3439}, abstract = {CRISPR/Cas-mediated editing has revolutionized crop engineering. Due to the broad scope and potential of this technology, many studies have been carried out in the past decade towards optimizing genome editing constructs. Clearly, the choice of the promoter used to drive gRNA and Cas9 expression is critical to achieving high editing efficiency, precision, and heritability. While some important considerations for choosing a promoter include the number and nature of targets, host organism, mode of transformation and goal of the experiment, spatiotemporal regulation of Cas9 expression using tissue-specific or inducible promoters enables higher heritability and efficiency of targeted mutagenesis with reduced off-target effects. In this review, we discuss specific studies that highlight the prospects and trade-offs associated with the choice of promoters on genome editing and emphasize the need for inductive exploration and discovery to further advance this area of research in crop plants.}, }
@article {pmid35557039, year = {2022}, author = {Ye, J and Xi, H and Chen, Y and Chen, Q and Lu, X and Lv, J and Chen, Y and Gu, F and Zhao, J}, title = {Can SpRY recognize any PAM in human cells?.}, journal = {Journal of Zhejiang University. Science. B}, volume = {23}, number = {5}, pages = {382-391}, doi = {10.1631/jzus.B2100710}, pmid = {35557039}, issn = {1862-1783}, support = {18331105//Lin HE's Academician Workstation of New Medicine and Clinical Translation/ ; H22010011//Program for Basic Science and Technology Cooperation Projects of Wenzhou City/ ; }, abstract = {The application of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) can be limited due to a lack of compatible protospacer adjacent motif (PAM) sequences in the DNA regions of interest. Recently, SpRY, a variant of Streptococcus pyogenes Cas9 (SpCas9), was reported, which nearly completely fulfils the PAM requirement. Meanwhile, PAMs for SpRY have not been well addressed. In our previous study, we developed the PAM Definition by Observable Sequence Excision (PAM-DOSE) and green fluorescent protein (GFP)‍-reporter systems to study PAMs in human cells. Herein, we endeavored to identify the PAMs of SpRY with these two methods. The results indicated that 5'-NRN-3', 5'-NTA-3', and 5'-NCK-3' could be considered as canonical PAMs. 5'-NCA-3' and 5'-NTK-3' may serve as non-priority PAMs. At the same time, PAM of 5'-NYC-3' is not recommended for human cells. These findings provide further insights into the application of SpRY for human genome editing.}, }
@article {pmid35552388, year = {2022}, author = {Vicencio, J and Sánchez-Bolaños, C and Moreno-Sánchez, I and Brena, D and Vejnar, CE and Kukhtar, D and Ruiz-López, M and Cots-Ponjoan, M and Rubio, A and Melero, NR and Crespo-Cuadrado, J and Carolis, C and Pérez-Pulido, AJ and Giráldez, AJ and Kleinstiver, BP and Cerón, J and Moreno-Mateos, MA}, title = {Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {2601}, pmid = {35552388}, issn = {2041-1723}, mesh = {Animals ; *CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Caenorhabditis elegans/genetics/metabolism ; *Gene Editing/methods ; RNA, Guide/genetics ; RNA, Messenger ; Zebrafish/genetics/metabolism ; }, abstract = {The requirement for Cas nucleases to recognize a specific PAM is a major restriction for genome editing. SpCas9 variants SpG and SpRY, recognizing NGN and NRN PAMs, respectively, have contributed to increase the number of editable genomic sites in cell cultures and plants. However, their use has not been demonstrated in animals. Here we study the nuclease activity of SpG and SpRY by targeting 40 sites in zebrafish and C. elegans. Delivered as mRNA-gRNA or ribonucleoprotein (RNP) complexes, SpG and SpRY were able to induce mutations in vivo, albeit at a lower rate than SpCas9 in equivalent formulations. This lower activity was overcome by optimizing mRNA-gRNA or RNP concentration, leading to mutagenesis at regions inaccessible to SpCas9. We also found that the CRISPRscan algorithm could help to predict SpG and SpRY targets with high activity in vivo. Finally, we applied SpG and SpRY to generate knock-ins by homology-directed repair. Altogether, our results expand the CRISPR-Cas targeting genomic landscape in animals.}, }
@article {pmid35551240, year = {2022}, author = {Zhang, D and Wang, G and Yu, X and Wei, T and Farbiak, L and Johnson, LT and Taylor, AM and Xu, J and Hong, Y and Zhu, H and Siegwart, DJ}, title = {Enhancing CRISPR/Cas gene editing through modulating cellular mechanical properties for cancer therapy.}, journal = {Nature nanotechnology}, volume = {}, number = {}, pages = {}, pmid = {35551240}, issn = {1748-3395}, support = {RP160157//Cancer Prevention and Research Institute of Texas (Cancer Prevention Research Institute of Texas)/ ; RP190251//Cancer Prevention and Research Institute of Texas (Cancer Prevention Research Institute of Texas)/ ; Predoctoral Fellowship in Drug Delivery//Pharmaceutical Research and Manufacturers of America Foundation (PhRMA Foundation)/ ; R01 CA251928/CA/NCI NIH HHS/United States ; 5P30CA142543//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; R01 CA269787-01//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; R01 EB025192-01A1//U.S. Department of Health &amp; Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)/ ; RSG-17-012-01//American Cancer Society (American Cancer Society, Inc.)/ ; I-1855//Welch Foundation/ ; SIEGWA18XX0//Cystic Fibrosis Foundation (CF Foundation)/ ; }, abstract = {Genome editing holds great potential for cancer treatment due to the ability to precisely inactivate or repair cancer-related genes. However, delivery of CRISPR/Cas to solid tumours for efficient cancer therapy remains challenging. Here we targeted tumour tissue mechanics via a multiplexed dendrimer lipid nanoparticle (LNP) approach involving co-delivery of focal adhesion kinase (FAK) siRNA, Cas9 mRNA and sgRNA (siFAK + CRISPR-LNPs) to enable tumour delivery and enhance gene-editing efficacy. We show that gene editing was enhanced >10-fold in tumour spheroids due to increased cellular uptake and tumour penetration of nanoparticles mediated by FAK-knockdown. siFAK + CRISPR-PD-L1-LNPs reduced extracellular matrix stiffness and efficiently disrupted PD-L1 expression by CRISPR/Cas gene editing, which significantly inhibited tumour growth and metastasis in four mouse models of cancer. Overall, we provide evidence that modulating the stiffness of tumour tissue can enhance gene editing in tumours, which offers a new strategy for synergistic LNPs and other nanoparticle systems to treat cancer using gene editing.}, }
@article {pmid35550915, year = {2022}, author = {Li, Y and Mensah, EO and Fordjour, E and Bai, J and Yang, Y and Bai, Z}, title = {Recent advances in high-throughput metabolic engineering: Generation of oligonucleotide-mediated genetic libraries.}, journal = {Biotechnology advances}, volume = {}, number = {}, pages = {107970}, doi = {10.1016/j.biotechadv.2022.107970}, pmid = {35550915}, issn = {1873-1899}, abstract = {The preparation of genetic libraries is an essential step to evolve microorganisms and study genotype-phenotype relationships by high-throughput screening/selection. As the large-scale synthesis of oligonucleotides becomes easy, cheap, and high-throughput, numerous novel strategies have been developed in recent years to construct high-quality oligo-mediated libraries, leveraging state-of-art molecular biology tools for genome editing and gene regulation. This review presents an overview of recent advances in creating and characterizing in vitro and in vivo genetic libraries, based on CRISPR/Cas, regulatory RNAs, and recombineering, primarily for Escherichia coli and Saccharomyces cerevisiae. These libraries' applications in high-throughput metabolic engineering, strain evolution and protein engineering are also discussed.}, }
@article {pmid35550024, year = {2022}, author = {Mesa, V and Monot, M and Ferraris, L and Popoff, M and Mazuet, C and Barbut, F and Delannoy, J and Dupuy, B and Butel, MJ and Aires, J}, title = {Core-, pan- and accessory genome analyses of Clostridium neonatale: insights into genetic diversity.}, journal = {Microbial genomics}, volume = {8}, number = {5}, pages = {}, doi = {10.1099/mgen.0.000813}, pmid = {35550024}, issn = {2057-5858}, mesh = {*Clostridium/genetics ; Genetic Variation ; *Genome, Bacterial ; Humans ; Infant, Newborn ; Phylogeny ; }, abstract = {Clostridium neonatale is a potential opportunistic pathogen recovered from faecal samples in cases of necrotizing enterocolitis (NEC), a gastrointestinal disease affecting preterm neonates. Although the C. neonatale species description and name validation were published in 2018, comparative genomics are lacking. In the present study, we provide the closed genome assembly of the C. neonatale ATCC BAA-265T (=250.09) reference strain with a manually curated functional annotation of the coding sequences. Pan-, core- and accessory genome analyses were performed using the complete 250.09 genome (4.7 Mb), three new assemblies (4.6-5.6 Mb), and five publicly available draft genome assemblies (4.6-4.7 Mb). The C. neonatale pan-genome contains 6840 genes, while the core-genome has 3387 genes. Pan-genome analysis revealed an 'open' state and genomic diversity. The strain-specific gene families ranged from five to 742 genes. Multiple mobile genetic elements were predicted, including a total of 201 genomic islands, 13 insertion sequence families, one CRISPR-Cas type I-B system and 15 predicted intact prophage signatures. Primary virulence classes including offensive, defensive, regulation of virulence-associated genes and non-specific virulence factors were identified. The presence of a tet(W/N/W) gene encoding a tetracycline resistance ribosomal protection protein and a 23S rRNA methyltransferase ermQ gene were identified in two different strains. Together, our results revealed a genetic diversity and plasticity of C. neonatale genomes and provide a comprehensive view of this species genomic features, paving the way for the characterization of its biological capabilities.}, }
@article {pmid35549895, year = {2022}, author = {Bernas, G and Ouellet, M and Barrios, A and Jamann, H and Larochelle, C and Lévy, É and Schmouth, JF}, title = {Introduction of loxP sites by electroporation in the mouse genome; a simple approach for conditional allele generation in complex targeting loci.}, journal = {BMC biotechnology}, volume = {22}, number = {1}, pages = {14}, pmid = {35549895}, issn = {1472-6750}, support = {EGID 3322//Multiple Sclerosis Society of Canada/ ; }, mesh = {Alleles ; Animals ; CRISPR-Cas Systems/genetics ; *Electroporation/methods ; Embryo, Mammalian ; Exons ; Mammals/genetics ; Mice ; *Zygote ; }, abstract = {BACKGROUND: The discovery of the CRISPR-Cas9 system and its applicability in mammalian embryos has revolutionized the way we generate genetically engineered animal models. To date, models harbouring conditional alleles (i.e. two loxP sites flanking an exon or a critical DNA sequence of interest) are amongst the most widely requested project type that are challenging to generate as they require simultaneous cleavage of the genome using two guides in order to properly integrate the repair template. An approach, using embryo sequential electroporation has been reported in the literature to successfully introduce loxP sites on the same allele. Here, we describe a modification of this sequential electroporation procedure that demonstrated the production of conditional allele mouse models for eight different genes via one of two possible strategies: either by consecutive sequential electroporation (strategy A) or non-consecutive sequential electroporation (strategy B). This latest strategy originated from using the by-product produced when using consecutive sequential electroporation (i.e. mice with a single targeted loxP site) to complete the project.<br><br>RESULTS: By using strategy A, we demonstrated successful generation of conditional allele models for three different genes (Icam1, Lox, and Sar1b), with targeting efficiencies varying between 5 and 13%. By using strategy B, we generated five conditional allele models (Loxl1, Pard6a, Pard6g, Clcf1, and Mapkapk5), with targeting efficiencies varying between 3 and 25%.<br><br>CONCLUSION: Our modified electroporation-based approach, involving one of the two alternative strategies, allowed the production of conditional allele models for eight different genes via two different possible paths. This reproducible method will serve as another reliable approach in addition to other well-established methodologies in the literature for conditional allele mouse model generation.}, }
@article {pmid35549133, year = {2021}, author = {Kanduri, V and LaVigne, D and Larsen, J}, title = {Current Advances Toward the Encapsulation of Cas9.}, journal = {ACS macro letters}, volume = {10}, number = {12}, pages = {1576-1589}, doi = {10.1021/acsmacrolett.1c00538}, pmid = {35549133}, issn = {2161-1653}, mesh = {*CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; Lipids ; Liposomes ; Nanoparticles ; Polymers/metabolism ; Quality of Life ; }, abstract = {Genetic diseases present formidable hurdles in maintaining a good quality of life for those suffering from these ailments. Often, patients look to inadequate treatments to manage symptoms, which can result in harmful effects on the body. Through genetic engineering, scientists utilize the clustered regularly short palindromic repeat (CRISPR)-associated protein, known as Cas9, to treat the root of the problem. The Cas9 protein is often codelivered with guide RNAs or in ribonucleoprotein complexes (RNP) to ensure targeted delivery of the genetic tool as well as to limit off-target effects. This paper provides an overview of the current advances made toward the encapsulation and delivery of Cas9 to desired locations in the body through encapsulating nanoparticles. Several factors must be considered when employing the Cas9 system to allow gene editing to occur. Material selection is crucial to protect the payload of the delivery vector. Current literature indicates that lipid- and polymer-based nanoparticles show the most potential as delivery vessels for Cas9. Lipid nanoparticles greatly outpace polymer-based nanoparticles in the clinic, despite the benefits that polymers may introduce. When developing translatable systems, there are factors that have not yet been considered that are relevant to Cas9 delivery that are highlighted in this Viewpoint. The proper functioning of Cas9 is dependent on maintaining a proper internal environment; however, there are gaps in the literature regarding these optimal conditions. Interactions between charges of the Cas9 protein, codelivered molecules, and delivery vehicles could impact the effectiveness of the gene editing taking place. While the internal charges of nanoparticles and their effects on Cas9 are presently undetermined, nanoparticles currently offer the ideal delivery method for the Cas9 protein due to their adequate size, modifiable external charge, and ability to be modified. Overall, a cationic lipid-/polymer-based nanoparticle system was found to have the most prospects in Cas9 delivery thus far. By understanding the successes of other systems, translatable, polymer-based delivery vehicles may be developed.}, }
@article {pmid35548699, year = {2022}, author = {Sturme, MHJ and van der Berg, JP and Bouwman, LMS and De Schrijver, A and de Maagd, RA and Kleter, GA and Battaglia-de Wilde, E}, title = {Occurrence and Nature of Off-Target Modifications by CRISPR-Cas Genome Editing in Plants.}, journal = {ACS agricultural science &amp; technology}, volume = {2}, number = {2}, pages = {192-201}, pmid = {35548699}, issn = {2692-1952}, abstract = {CRISPR-Cas-based genome editing allows for precise and targeted genetic modification of plants. Nevertheless, unintended off-target edits can arise that might confer risks when present in gene-edited food crops. Through an extensive literature review we gathered information on CRISPR-Cas off-target edits in plants. Most observed off-target changes were small insertions or deletions (1-22 bp) or nucleotide substitutions, and large deletions (>100 bp) were rare. One study detected the insertion of vector-derived DNA sequences, which is important considering the risk assessment of gene-edited plants. Off-target sites had few mismatches (1-3 nt) with the target sequence and were mainly located in protein-coding regions, often in target gene homologues. Off-targets edits were predominantly detected via biased analysis of predicted off-target sites instead of unbiased genome-wide analysis. CRISPR-Cas-edited plants showed lower off-target mutation frequencies than conventionally bred plants. This Review can aid discussions on the relevance of evaluating off-target modifications for risk assessment of CRISPR-Cas-edited plants.}, }
@article {pmid35547744, year = {2022}, author = {Chan, YT and Lu, Y and Wu, J and Zhang, C and Tan, HY and Bian, ZX and Wang, N and Feng, Y}, title = {CRISPR-Cas9 library screening approach for anti-cancer drug discovery: overview and perspectives.}, journal = {Theranostics}, volume = {12}, number = {7}, pages = {3329-3344}, pmid = {35547744}, issn = {1838-7640}, mesh = {*Antineoplastic Agents/pharmacology/therapeutic use ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genetic Engineering ; Humans ; *Neoplasms/drug therapy/genetics ; }, abstract = {CRISPR-Cas9 is a Nobel Prize-winning robust gene-editing tool developed in the last decade. This technique enables a stable genetic engineering method with high precision on the genomes of all organisms. The latest advances in the technology include a genome library screening approach, which can detect survival-essential and drug resistance genes via gain or loss of function. The versatile machinery allows genomic screening for gene activation or inhibition, and targets non-coding sequences, such as promoters, miRNAs, and lncRNAs. In this review, we introduce the emerging high-throughput CRISPR-Cas9 library genome screening technology and its working principles to detect survival and drug resistance genes through positive and negative selection. The technology is compared with other existing approaches while focusing on the advantages of its variable applications in anti-cancer drug discovery, including functions and target identification, non-coding RNA information, actions of small molecules, and drug target discoveries. The combination of the CRISPR-Cas9 system with multi-omic platforms represents a dynamic field expected to advance anti-cancer drug discovery and precision medicine in the clinic.}, }
@article {pmid35544771, year = {2022}, author = {Liu, W and An, C and Shu, X and Meng, X and Yao, Y and Zhang, J and Chen, F and Xiang, H and Yang, S and Gao, X and Gao, SS}, title = {Correction to "A Dual-Plasmid CRISPR/Cas System for Mycotoxin Elimination in Polykaryotic Industrial Fungi".}, journal = {ACS synthetic biology}, volume = {}, number = {}, pages = {}, doi = {10.1021/acssynbio.2c00228}, pmid = {35544771}, issn = {2161-5063}, }
@article {pmid35543560, year = {2022}, author = {Li, Y and Zhang, L and Yang, H and Xia, Y and Liu, L and Chen, X and Shen, W}, title = {Development of a gRNA Expression and Processing Platform for Efficient CRISPR-Cas9-Based Gene Editing and Gene Silencing in Candida tropicalis.}, journal = {Microbiology spectrum}, volume = {}, number = {}, pages = {e0005922}, doi = {10.1128/spectrum.00059-22}, pmid = {35543560}, issn = {2165-0497}, abstract = {Candida tropicalis, a nonmodel diploid microbe, has been applied in industry as a chassis cell. Metabolic engineering of C. tropicalis is challenging due to a lack of gene editing and regulation tools. Here, we report a tRNA:guide RNA (gRNA) platform for boosting gene editing and silencing efficiency in C. tropicalis. As the endogenous tRNA-processing system enables autocleavage for producing a large number of mature gRNAs, a tRNAGly sequence from the genome of C. tropicalis ATCC 20336 was selected for constructing the tRNA:gRNA platform. In the CRISPR-Cas9 system, the tRNA:gRNA platform proved to be efficient in single-gene and multi-gene editing. Furthermore, based on the tRNA:gRNA platform, a CRISPR interference (CRISPRi) system was developed to construct an efficient dCas9-mediated gene expression regulation system for C. tropicalis. The CRISPRi system was employed to regulate the expression of the exogenous gene GFP3 (green fluorescent protein) and the endogenous gene ADE2 (phosphoribosylaminoimidazole carboxylase). Different regions of GFP3 and ADE2 were targeted with the gRNAs processed by the tRNAGly, and the transcription levels of GFP3 and ADE2 were successfully downregulated to 23.9% ± 4.1% and 38.0% ± 7.4%, respectively. The effects of the target regions on gene regulation were also investigated. Additionally, the regulation system was applied to silence ERG9 (squalene synthase) to enhance β-carotene biosynthesis in a metabolically modified C. tropicalis strain. The results suggest that the endogenous tRNAGly and the CRISPRi system have great potential for metabolic engineering of C. tropicalis. IMPORTANCE In the nonmodel yeast Candida tropicalis, a lack of available RNA polymerase type III (Pol III) promoters hindered the development of guide RNA (gRNA) expression platforms for the establishment of CRISPR-Cas-mediated genome editing and silencing strategies. Here, a tRNA:gRNA platform was constructed. We show that this platform allows efficient and precise expression and processing of different gRNAs from a single polycistronic gene capable of mediating multi-gene editing in combination with CRISPR-Cas9. Furthermore, in combination with dCas9, the tRNA:gRNA platform was efficiently used for silencing of exogenous and endogenous genes, representing the first CRISPR interference tool (CRISPRi) in C. tropicalis. Importantly, the established CRISPRi-tRNA:gRNA tool was also used for metabolic engineering by regulating β-carotene biosynthesis in C. tropicalis. The results suggest that the tRNA:gRNA platform and the CRISPRi system will further advance the application of the CRISPR-Cas-based editing and CRISPRi systems for metabolic engineering in C. tropicalis.}, }
@article {pmid35538629, year = {2022}, author = {Bellingrath, JS and McClements, ME and Shanks, M and Clouston, P and Fischer, MD and MacLaren, RE}, title = {Envisioning the development of a CRISPR-Cas mediated base editing strategy for a patient with a novel pathogenic CRB1 single nucleotide variant.}, journal = {Ophthalmic genetics}, volume = {}, number = {}, pages = {1-10}, doi = {10.1080/13816810.2022.2073599}, pmid = {35538629}, issn = {1744-5094}, abstract = {BACKGROUND: Inherited retinal degeneration (IRD) associated with mutations in the Crumbs homolog 1 (CRB1) gene is associated with a severe, early-onset retinal degeneration for which no therapy currently exists. Base editing, with its capability to precisely catalyse permanent nucleobase conversion in a programmable manner, represents a novel therapeutic approach to targeting this autosomal recessive IRD, for which a gene supplementation is challenging due to the need to target three different retinal CRB1 isoforms.<br><br>PURPOSE: To report and classify a novel CRB1 variant and envision a possible therapeutic approach in form of base editing.<br><br>METHODS: Case report.<br><br>RESULTS: A 16-year-old male patient with a clinical diagnosis of early-onset retinitis pigmentosa (RP) and characteristic clinical findings of retinal thickening and coarse lamination was seen at the Oxford Eye Hospital. He was found to be compound heterozygous for two CRB1 variants: a novel pathogenic nonsense variant in exon 9, c.2885T>A (p.Leu962Ter), and a likely pathogenic missense change in exon 6, c.2056C>T (p.Arg686Cys). While a base editing strategy for c.2885T>A would encompass a CRISPR-pass mediated "read-through" of the premature stop codon, the resulting missense changes were predicted to be "possibly damaging" in in-silico analysis. On the other hand, the transversion missense change, c.2056C>T, is amenable to transition editing with an adenine base editor (ABE) fused to a SaCas9-KKH with a negligible chance of bystander edits due to an absence of additional Adenines (As) in the editing window.<br><br>CONCLUSIONS: This case report records a novel pathogenic nonsense variant in CRB1 and gives an example of thinking about a base editing strategy for a patient compound heterozygous for CRB1 variants.}, }
@article {pmid35538076, year = {2022}, author = {Dong, C and Fu, S and Karvas, RM and Chew, B and Fischer, LA and Xing, X and Harrison, JK and Popli, P and Kommagani, R and Wang, T and Zhang, B and Theunissen, TW}, title = {A genome-wide CRISPR-Cas9 knockout screen identifies essential and growth-restricting genes in human trophoblast stem cells.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {2548}, pmid = {35538076}, issn = {2041-1723}, mesh = {CRISPR-Cas Systems ; Cell Differentiation/genetics ; DNA-Binding Proteins/genetics/metabolism ; Female ; Humans ; *Placenta/metabolism ; Pregnancy ; Protein Tyrosine Phosphatases, Non-Receptor/genetics ; Stem Cells/metabolism ; Transcription Factors/genetics/metabolism ; *Trophoblasts/metabolism ; }, abstract = {The recent derivation of human trophoblast stem cells (hTSCs) provides a scalable in vitro model system of human placental development, but the molecular regulators of hTSC identity have not been systematically explored thus far. Here, we utilize a genome-wide CRISPR-Cas9 knockout screen to comprehensively identify essential and growth-restricting genes in hTSCs. By cross-referencing our data to those from similar genetic screens performed in other cell types, as well as gene expression data from early human embryos, we define hTSC-specific and -enriched regulators. These include both well-established and previously uncharacterized trophoblast regulators, such as ARID3A, GATA2, and TEAD1 (essential), and GCM1, PTPN14, and TET2 (growth-restricting). Integrated analysis of chromatin accessibility, gene expression, and genome-wide location data reveals that the transcription factor TEAD1 regulates the expression of many trophoblast regulators in hTSCs. In the absence of TEAD1, hTSCs fail to complete faithful differentiation into extravillous trophoblast (EVT) cells and instead show a bias towards syncytiotrophoblast (STB) differentiation, thus indicating that this transcription factor safeguards the bipotent lineage potential of hTSCs. Overall, our study provides a valuable resource for dissecting the molecular regulation of human placental development and diseases.}, }
@article {pmid35536747, year = {2022}, author = {Gotoh, Y and Atsuta, Y and Taniguchi, T and Nishida, R and Nakamura, K and Ogura, Y and Misawa, N and Hayashi, T}, title = {Helicobacter cinaedi is a human-adapted lineage in the Helicobacter cinaedi/canicola/'magdeburgensis' complex.}, journal = {Microbial genomics}, volume = {8}, number = {5}, pages = {}, doi = {10.1099/mgen.0.000830}, pmid = {35536747}, issn = {2057-5858}, mesh = {Animals ; *Bacteremia ; Cricetinae ; Dogs ; *Helicobacter/genetics ; *Helicobacter Infections ; Humans ; Rats ; }, abstract = {Helicobacter cinaedi is an enterohepatic Helicobacter that causes bacteremia and other diseases in humans. While H. cinaedi-like strains are isolated from animals, including dog isolates belonging to a recently proposed H. canicola, little is known about the genetic differences between H. cinaedi and these animal isolates. Here, we sequenced 43 H. cinaedi- or H. canicola-like strains isolated from humans, hamsters, rats and dogs and collected 81 genome sequences of H. cinaedi, H. canicola and other enterohepatic Helicobacter strains from public databases. Genomic comparison of these strains identified four distinct clades (clades I-IV) in H. cinaedi/canicola/'magderbugensis' (HCCM) complex. Among these, clade I corresponds to H. cinaedi sensu stricto and represents a human-adapted lineage in the complex. We identified several genomic features unique to clade I. They include the accumulation of antimicrobial resistance-related mutations that reflects the human association of clade I and the larger genome size and the presence of a CRISPR-Cas system and multiple toxin-antitoxin and restriction-modification systems, both of which indicate the contribution of horizontal gene transfer to the evolution of clade I. In addition, nearly all clade I strains but only a few strains belonging to one minor clade contained a highly variable genomic region encoding a type VI secretion system (T6SS), which could play important roles in gut colonization by killing competitors or inhibiting their growth. We also developed a method to systematically search for H. cinaedi sequences in large metagenome data sets based on the results of genome comparison. Using this method, we successfully identified multiple HCCM complex-containing human faecal metagenome samples and obtained the sequence information covering almost the entire genome of each strain. Importantly, all were clade I strains, supporting our conclusion that H. cinaedi sensu stricto is a human-adapted lineage in the HCCM complex.}, }
@article {pmid35534475, year = {2022}, author = {Bishop, AL and López Del Amo, V and Okamoto, EM and Bodai, Z and Komor, AC and Gantz, VM}, title = {Double-tap gene drive uses iterative genome targeting to help overcome resistance alleles.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {2595}, pmid = {35534475}, issn = {2041-1723}, support = {MCB-2048207//National Science Foundation (NSF)/ ; DP5OD023098//U.S. Department of Health &amp; Human Services | NIH | NIH Office of the Director (OD)/ ; R01AI162911//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)/ ; }, mesh = {Alleles ; Animals ; CRISPR-Cas Systems/genetics ; *Gene Drive Technology ; Germ Cells ; Mammals/genetics ; Mice ; RNA, Guide/genetics ; }, abstract = {Homing CRISPR gene drives could aid in curbing the spread of vector-borne diseases and controlling crop pest and invasive species populations due to an inheritance rate that surpasses Mendelian laws. However, this technology suffers from resistance alleles formed when the drive-induced DNA break is repaired by error-prone pathways, which creates mutations that disrupt the gRNA recognition sequence and prevent further gene-drive propagation. Here, we attempt to counteract this by encoding additional gRNAs that target the most commonly generated resistance alleles into the gene drive, allowing a second opportunity at gene-drive conversion. Our presented "double-tap" strategy improved drive efficiency by recycling resistance alleles. The double-tap drive also efficiently spreads in caged populations, outperforming the control drive. Overall, this double-tap strategy can be readily implemented in any CRISPR-based gene drive to improve performance, and similar approaches could benefit other systems suffering from low HDR frequencies, such as mammalian cells or mouse germline transformations.}, }
@article {pmid35534455, year = {2022}, author = {Bodai, Z and Bishop, AL and Gantz, VM and Komor, AC}, title = {Targeting double-strand break indel byproducts with secondary guide RNAs improves Cas9 HDR-mediated genome editing efficiencies.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {2351}, pmid = {35534455}, issn = {2041-1723}, support = {DP5OD023098//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)/ ; MCB-2048207//National Science Foundation (NSF)/ ; }, mesh = {Animals ; CRISPR-Cas Systems/genetics ; DNA End-Joining Repair ; *Gene Editing/methods ; Mammals/genetics ; *RNA, Guide/genetics/metabolism ; Recombinational DNA Repair ; }, abstract = {Programmable double-strand DNA breaks (DSBs) can be harnessed for precision genome editing through manipulation of the homology-directed repair (HDR) pathway. However, end-joining repair pathways often outcompete HDR and introduce insertions and deletions of bases (indels) at the DSB site, decreasing precision outcomes. It has been shown that indel sequences for a given DSB site are reproducible and can even be predicted. Here, we report a general strategy (the "double tap" method) to improve HDR-mediated precision genome editing efficiencies that takes advantage of the reproducible nature of indel sequences. The method simply involves the use of multiple gRNAs: a primary gRNA that targets the wild-type genomic sequence, and one or more secondary gRNAs that target the most common indel sequence(s), which in effect provides a "second chance" at HDR-mediated editing. This proof-of-principle study presents the double tap method as a simple yet effective option for enhancing precision editing in mammalian cells.}, }
@article {pmid35532260, year = {2022}, author = {Chambers, C and Quan, L and Yi, G and Esquela-Kerscher, A}, title = {CRISPR Gene Editing Tool for MicroRNA Cluster Network Analysis.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {182}, pages = {}, doi = {10.3791/63704}, pmid = {35532260}, issn = {1940-087X}, mesh = {CRISPR-Cas Systems ; Endonucleases/genetics ; *Gene Editing/methods ; Humans ; *MicroRNAs/genetics ; RNA, Guide/genetics ; RNA, Untranslated ; }, abstract = {MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published studies focus on a single miRNA when characterizing the role of small RNAs in cancer. However, ~30% of human miRNA genes are organized in clustered units that are often co-expressed, indicating a complex and coordinated system of noncoding RNA regulation. A clearer understating of how clustered miRNA networks function cooperatively to regulate tumor growth, cancer aggressiveness, and drug resistance is required before translating noncoding small RNAs to the clinic. The use of a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene editing procedure has been employed to study the oncogenic role of a genomic cluster of seven miRNA genes located within a locus spanning ~35,000 bp in length in the context of prostate cancer. For this approach, human cancer cell lines were infected with a lentivirus vector for doxycycline (DOX)-inducible Cas9 nuclease grown in DOX-containing medium for 48 h. The cells were subsequently co-transfected with synthetic trans-activating CRISPR RNA (tracrRNA) complexed with genomic site-specific CRISPR RNA (crRNA) oligonucleotides to allow the rapid generation of cancer cell lines carrying the entire miRNA cluster deletion and individual or combination miRNA gene cluster deletions within a single experiment. The advantages of this high-throughput gene editing system are the ability to avoid time-consuming DNA vector subcloning, the flexibility in transfecting cells with unique guide RNA combinations in a 24-well format, and the lower-cost PCR genotyping using crude cell lysates. Studies using this streamlined approach promise to uncover functional redundancies and synergistic/antagonistic interactions between miRNA cluster members, which will aid in characterizing the complex small noncoding RNA networks involved in human disease and better inform future therapeutic design.}, }
@article {pmid35531207, year = {2022}, author = {Raza, SHA and Hassanin, AA and Pant, SD and Bing, S and Sitohy, MZ and Abdelnour, SA and Alotaibi, MA and Al-Hazani, TM and Abd El-Aziz, AH and Cheng, G and Zan, L}, title = {Potentials, prospects and applications of genome editing technologies in livestock production.}, journal = {Saudi journal of biological sciences}, volume = {29}, number = {4}, pages = {1928-1935}, pmid = {35531207}, issn = {1319-562X}, abstract = {In recent years, significant progress has been achieved in genome editing applications using new programmable DNA nucleases such as zinc finger nucleases (ZFNs), transcription activator-like endonucleases (TALENs) and the clustered regularly interspaced short palindromic repeats/Cas9 system (CRISPR/Cas9). These genome editing tools are capable of nicking DNA precisely by targeting specific sequences, and enable the addition, removal or substitution of nucleotides via double-stranded breakage at specific genomic loci. CRISPR/Cas system, one of the most recent genome editing tools, affords the ability to efficiently generate multiple genomic nicks in single experiment. Moreover, CRISPR/Cas systems are relatively easy and cost effective when compared to other genome editing technologies. This is in part because CRISPR/Cas systems rely on RNA-DNA binding, unlike other genome editing tools that rely on protein-DNA interactions, which affords CRISPR/Cas systems higher flexibility and more fidelity. Genome editing tools have significantly contributed to different aspects of livestock production such as disease resistance, improved performance, alterations of milk composition, animal welfare and biomedicine. However, despite these contributions and future potential, genome editing technologies also have inherent risks, and therefore, ethics and social acceptance are crucial factors associated with implementation of these technologies. This review emphasizes the impact of genome editing technologies in development of livestock breeding and production in numerous species such as cattle, pigs, sheep and goats. This review also discusses the mechanisms behind genome editing technologies, their potential applications, risks and associated ethics that should be considered in the context of livestock.}, }
@article {pmid35525561, year = {2022}, author = {Cassidy, AM and Kuliyev, E and Thomas, DB and Chen, H and Pelletier, S}, title = {Dissecting protein function in vivo: Engineering allelic series in mice using CRISPR-Cas9 technology.}, journal = {Methods in enzymology}, volume = {667}, number = {}, pages = {775-812}, doi = {10.1016/bs.mie.2022.03.053}, pmid = {35525561}, issn = {1557-7988}, mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Gene Targeting ; Mice ; Mutagenesis ; Technology ; }, abstract = {Allelic series are extremely valuable genetic tools to study gene function and identify essential structural features of gene products. In mice, allelic series have been engineered using conventional gene targeting in embryonic stem cells or chemical mutagenesis. While these approaches have provided valuable information about the function of genes, they remain cumbersome. Modern approaches such as CRISPR-Cas9 technologies now allow for the precise and cost-effective generation of mouse models with specific mutations, facilitating the development of allelic series. Here, we describe procedures for the generation of three types of mutations used to dissect protein function in vivo using CRISPR-Cas9 technology. This step-by-step protocol describes the generation of missense mutations, large in-frame deletions, and insertions of genetic material using SCY1-like 1 (Scyl1) as a model gene.}, }
@article {pmid35525543, year = {2022}, author = {Jacobsen, AV and Murphy, JM}, title = {CRISPR deletions in cell lines for reconstitution studies of pseudokinase function.}, journal = {Methods in enzymology}, volume = {667}, number = {}, pages = {229-273}, doi = {10.1016/bs.mie.2022.03.054}, pmid = {35525543}, issn = {1557-7988}, mesh = {*CRISPR-Cas Systems ; Cell Line ; *Gene Editing/methods ; Humans ; *Protein Kinases/genetics ; }, abstract = {The non-catalytic cousins of protein kinases, the pseudokinases, have grown to prominence as indispensable signaling entities over the past decade, despite their lack of catalytic activity. Because their importance has only been fully embraced recently, many of the 10% of the human kinome categorized as pseudokinases are yet to be attributed biological functions. The advent of CRISPR-Cas9 editing to genetically delete pseudokinases in a cell line of interest has proven invaluable to dissecting many functions and remains the method of choice for gene knockout. Here, using the terminal effector pseudokinase in the necroptosis cell death pathway, MLKL, as an exemplar, we describe a method for genetic knockout of pseudokinases in cultured cells. This method does not retain the CRISPR guide sequence in the edited cells, which eliminates possible interference in subsequent reconstitution studies where mutant forms of the pseudokinase can be reintroduced into cells exogenously for detailed mechanistic characterization.}, }
@article {pmid35524183, year = {2022}, author = {Zhou, S and Kalds, P and Luo, Q and Sun, K and Zhao, X and Gao, Y and Cai, B and Huang, S and Kou, Q and Petersen, B and Chen, Y and Ma, B and Wang, X}, title = {Optimized Cas9:sgRNA delivery efficiently generates biallelic MSTN knockout sheep without affecting meat quality.}, journal = {BMC genomics}, volume = {23}, number = {1}, pages = {348}, pmid = {35524183}, issn = {1471-2164}, support = {31872332//National Natural Science Foundation of China/ ; 31972526//National Natural Science Foundation of China/ ; NXTS2021-001//Local Grant/ ; 2021YFF1000700//National Key Research and Development Program of China/ ; }, mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/methods ; Goats/genetics ; Meat ; *Myostatin/genetics ; RNA, Guide/genetics ; RNA, Messenger ; Sheep/genetics ; }, abstract = {BACKGROUND: CRISPR/Cas9-based genome-editing systems have been used to efficiently engineer livestock species with precise genetic alterations intended for biomedical and agricultural applications. Previously, we have successfully generated gene-edited sheep and goats via one-cell-stage embryonic microinjection of a Cas9 mRNA and single-guide RNAs (sgRNAs) mixture. However, most gene-edited animals produced using this approach were heterozygotes. Additionally, non-homozygous gene-editing outcomes may not fully generate the desired phenotype in an efficient manner.<br><br>RESULTS: We report the optimization of a Cas9 mRNA-sgRNA delivery system to efficiently generate homozygous myostatin (MSTN) knockout sheep for improved growth and meat production. Firstly, an sgRNA selection software (sgRNAcas9) was used to preliminarily screen for highly efficient sgRNAs. Ten sgRNAs targeting the MSTN gene were selected and validated in vitro using sheep fibroblast cells. Four out of ten sgRNAs (two in exon 1 and two in exon 2) showed a targeting efficiency > 50%. To determine the optimal CRISPR/Cas9 microinjection concentration, four levels of Cas9 mRNA and three levels of sgRNAs in mixtures were injected into sheep embryos. Microinjection of 100 ng/μL Cas9 mRNA and 200 ng/μL sgRNAs resulted in the most improved targeting efficiency. Additionally, using both the highly efficient sgRNAs and the optimal microinjection concentration, MSTN-knockout sheep were generated with approximately 50% targeting efficiency, reaching a homozygous knockout efficiency of 25%. Growth rate and meat quality of MSTN-edited lambs were also investigated. MSTN-knockout lambs exhibited increased body weight and average daily gain. Moreover, pH, drip loss, intramuscular fat, crude protein, and shear force of gluteal muscles of MSTN-knockout lambs did not show changes compared to the wild-type lambs.<br><br>CONCLUSIONS: This study highlights the importance of in vitro evaluation for the optimization of sgRNAs and microinjection dosage of gene editing reagents. This approach enabled efficient engineering of homozygous knockout sheep. Additionally, this study confirms that MSTN-knockout lambs does not negatively impact meat quality, thus supporting the adoption of gene editing as tool to improve productivity of farm animals.}, }
@article {pmid35524126, year = {2022}, author = {Després, PC and Dubé, AK and Yachie, N and Landry, CR}, title = {High-Throughput Gene Mutagenesis Screening Using Base Editing.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2477}, number = {}, pages = {331-348}, pmid = {35524126}, issn = {1940-6029}, mesh = {Base Sequence ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Mutagenesis/genetics ; RNA, Guide/genetics ; }, abstract = {Base editing is a CRISPR-Cas9 genome engineering tool that allows programmable mutagenesis without the creation of double-stranded breaks. Here, we describe the design and execution of large-scale base editing screens using the Target-AID base editor in yeast. Using this approach, thousands of sites can be mutated simultaneously. The effects of these mutations on fitness can be measured using a pooled growth competition assay followed by DNA sequencing of gRNAs as barcodes.}, }
@article {pmid35524121, year = {2022}, author = {Dubé, AK and Dandage, R and Dibyachintan, S and Dionne, U and Després, PC and Landry, CR}, title = {Deep Mutational Scanning of Protein-Protein Interactions Between Partners Expressed from Their Endogenous Loci In Vivo.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2477}, number = {}, pages = {237-259}, pmid = {35524121}, issn = {1940-6029}, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Mutation ; Point Mutation ; }, abstract = {Deep mutational scanning (DMS) generates mutants of a protein of interest in a comprehensive manner. CRISPR-Cas9 technology enables large-scale genome editing with high efficiency. Using both DMS and CRISPR-Cas9 therefore allows us to investigate the effects of thousands of mutations inserted directly in the genome. Combined with protein-fragment complementation assay (PCA), which enables the quantitative measurement of protein-protein interactions (PPIs) in vivo, these methods allow for the systematic assessment of the effects of mutations on PPIs in living cells. Here, we describe a method leveraging DMS, CRISPR-Cas9, and PCA to study the effect of point mutations on PPIs mediated by protein domains in yeast.}, }
@article {pmid35524052, year = {2022}, author = {Chhun, A and Alberti, F}, title = {CRISPR/Cas9-Based Methods for Inactivating Actinobacterial Biosynthetic Genes and Elucidating Function.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2489}, number = {}, pages = {201-222}, pmid = {35524052}, issn = {1940-6029}, mesh = {*Actinobacteria/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Streptomyces/genetics/metabolism ; }, abstract = {The CRISPR/Cas9 technology allows fast and marker-less genome engineering that can be employed to study secondary metabolism in actinobacteria. Here, we report a standard experimental protocol for the deletion of a biosynthetic gene in a Streptomyces species, using the vector pCRISPomyces-2 developed by Huimin Zhao and collaborators. We also describe how carrying out metabolite analysis can reveal the putative biosynthetic function of the inactivated gene.}, }
@article {pmid35524051, year = {2022}, author = {Massicard, JM and Su, L and Jacob, C and Weissman, KJ}, title = {Engineering Modular Polyketide Biosynthesis in Streptomyces Using CRISPR/Cas: A Practical Guide.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2489}, number = {}, pages = {173-200}, pmid = {35524051}, issn = {1940-6029}, mesh = {Animals ; Biosynthetic Pathways/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; *Polyketides/metabolism ; RNA, Guide/genetics/metabolism ; *Streptomyces/genetics/metabolism ; }, abstract = {The CRISPR/Cas system, which has been widely applied to organisms ranging from microbes to animals, is currently being adapted for use in Streptomyces bacteria. In this case, it is notably applied to rationally modify the biosynthetic pathways giving rise to the polyketide natural products, which are heavily exploited in the medical and agricultural arenas. Our aim here is to provide the potential user with a practical guide to exploit this approach for manipulating polyketide biosynthesis, by treating key experimental aspects including vector choice, design of the basic engineering components, and trouble-shooting.}, }
@article {pmid35522691, year = {2022}, author = {Matozel, EK and Parziale, S and Price, AC}, title = {A programmable DNA roadblock system using dCas9 and multivalent target sites.}, journal = {PloS one}, volume = {17}, number = {5}, pages = {e0268099}, pmid = {35522691}, issn = {1932-6203}, mesh = {Binding Sites ; CRISPR-Cas Systems ; *DNA/metabolism ; DNA-Binding Proteins/genetics ; *Endonucleases/metabolism ; RNA, Guide/genetics ; }, abstract = {A protein roadblock forms when a protein binds DNA and hinders translocation of other DNA binding proteins. These roadblocks can have significant effects on gene expression and regulation as well as DNA binding. Experimental methods for studying the effects of such roadblocks often target endogenous sites or introduce non-variable specific sites into DNAs to create binding sites for artificially introduced protein roadblocks. In this work, we describe a method to create programmable roadblocks using dCas9, a cleavage deficient mutant of the CRISPR effector nuclease Cas9. The programmability allows us to custom design target sites in a synthetic gene intended for in vitro studies. These target sites can be coded with multivalency-in our case, internal restriction sites which can be used in validation studies to verify complete binding of the roadblock. We provide full protocols and sequences and demonstrate how to use the internal restriction sites to verify complete binding of the roadblock. We also provide example results of the effect of DNA roadblocks on the translocation of the restriction endonuclease NdeI, which searches for its cognate site using one dimensional diffusion along DNA.}, }
@article {pmid35521548, year = {2022}, author = {Shor, O and Rabinowitz, R and Offen, D and Benninger, F}, title = {Computational normal mode analysis accurately replicates the activity and specificity profiles of CRISPR-Cas9 and high-fidelity variants.}, journal = {Computational and structural biotechnology journal}, volume = {20}, number = {}, pages = {2013-2019}, pmid = {35521548}, issn = {2001-0370}, abstract = {The CRISPR-Cas system has transformed the field of gene-editing and created opportunities for novel genome engineering therapeutics. The field has significantly progressed, and recently, CRISPR-Cas9 was utilized in clinical trials to target disease-causing mutations. Existing tools aim to predict the on-target efficacy and potential genome-wide off-targets by scoring a particular gRNA according to an array of gRNA design principles or machine learning algorithms based on empirical results of large numbers of gRNAs. However, such tools are unable to predict the editing outcome by variant Cas enzymes and can only assess potential off-targets related to reference genomes. Here, we employ normal mode analysis (NMA) to investigate the structure of the Cas9 protein complexed with its gRNA and target DNA and explore the function of the protein. Our results demonstrate the feasibility and validity of NMA to predict the activity and specificity of SpyCas9 in the presence of mismatches by comparison to empirical data. Furthermore, despite the absence of their exact structures, this method accurately predicts the enzymatic activity of known high-fidelity engineered Cas9 variants.}, }
@article {pmid35513429, year = {2022}, author = {Li, R and Klingbeil, O and Monducci, D and Young, MJ and Rodriguez, DJ and Bayyat, Z and Dempster, JM and Kesar, D and Yang, X and Zamanighomi, M and Vakoc, CR and Ito, T and Sellers, WR}, title = {Comparative optimization of combinatorial CRISPR screens.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {2469}, pmid = {35513429}, issn = {2041-1723}, support = {W81XWH-19-1-0271//U.S. Department of Defense (United States Department of Defense)/ ; 500506//Ludwig Institute for Cancer Research (Ludwig Cancer Research)/ ; }, mesh = {*Acidaminococcus/genetics ; *CRISPR-Cas Systems/genetics ; RNA, Guide/genetics ; Staphylococcus aureus/genetics ; Streptococcus pyogenes/genetics ; }, abstract = {Combinatorial CRISPR technologies have emerged as a transformative approach to systematically probe genetic interactions and dependencies of redundant gene pairs. However, the performance of different functional genomic tools for multiplexing sgRNAs vary widely. Here, we generate and benchmark ten distinct pooled combinatorial CRISPR libraries targeting paralog pairs to optimize digenic knockout screens. Libraries composed of dual Streptococcus pyogenes Cas9 (spCas9), orthogonal spCas9 and Staphylococcus aureus (saCas9), and enhanced Cas12a from Acidaminococcus were evaluated. We demonstrate a combination of alternative tracrRNA sequences from spCas9 consistently show superior effect size and positional balance between the sgRNAs as a robust combinatorial approach to profile genetic interactions of multiple genes.}, }
@article {pmid35512092, year = {2022}, author = {Taylor, JH and Walton, JC and McCann, KE and Norvelle, A and Liu, Q and Vander Velden, JW and Borland, JM and Hart, M and Jin, C and Huhman, KL and Cox, DN and Albers, HE}, title = {CRISPR-Cas9 editing of the arginine-vasopressin V1a receptor produces paradoxical changes in social behavior in Syrian hamsters.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {119}, number = {19}, pages = {e2121037119}, doi = {10.1073/pnas.2121037119}, pmid = {35512092}, issn = {1091-6490}, support = {IOS-1035960//National Science Foundation (NSF)/ ; MH109302/MH/NIMH NIH HHS/United States ; MH122622/MH/NIMH NIH HHS/United States ; }, mesh = {Aggression/physiology ; Animals ; Arginine/metabolism ; Arginine Vasopressin/genetics ; *CRISPR-Cas Systems ; Cricetinae ; Mesocricetus ; *Receptors, Vasopressin/genetics/metabolism ; Social Behavior ; }, abstract = {SignificanceArginine-vasopressin (AVP) acting on V1a receptors (Avpr1as) represents a key signaling mechanism in a brain circuit that increases the expression of social communication and aggression. We produced Syrian hamsters that completely lack Avpr1as (Avpr1a knockout [KO] hamsters) using the CRISPR-Cas9 system to more fully examine the role of Avpr1a in the expression of social behaviors. We confirmed the absence of Avpr1as in these hamsters by demonstrating 1) a complete lack of Avpr1a-specific receptor binding throughout the brain, 2) a behavioral insensitivity to centrally administered AVP, and 3) an absence of the well-known blood-pressure response produced by activating Avpr1as. Unexpectedly, however, Avpr1a KO hamsters displayed more social communication behavior and aggression toward same-sex conspecifics than did their wild-type (WT) littermates.}, }
@article {pmid35509467, year = {2022}, author = {Wang, DX and Wang, YX and Wang, J and Ma, JY and Liu, B and Tang, AN and Kong, DM}, title = {MnO2 nanosheets as a carrier and accelerator for improved live-cell biosensing application of CRISPR/Cas12a.}, journal = {Chemical science}, volume = {13}, number = {15}, pages = {4364-4371}, pmid = {35509467}, issn = {2041-6520}, abstract = {Besides gene-editing, the CRISPR/Cas12a system has also been widely used in in vitro biosensing, but its applications in live-cell biosensing are rare. One reason is lacking appropriate carriers to synchronously deliver all components of the CRISPR/Cas12a system into living cells. Herein, we demonstrate that MnO2 nanosheets are an excellent carrier of CRISPR/Cas12a due to the two important roles played by them. Through a simple mixing operation, all components of the CRISPR/Cas12a system can be loaded on MnO2 nanosheets and thus synchronously delivered into cells. Intracellular glutathione (GSH)-induced decomposition of MnO2 nanosheets not only results in the rapid release of the CRISPR/Cas12a system in cells but also provides Mn2+ as an accelerator to promote CRISPR/Cas12a-based biosensing of intracellular targets. Due to the merits of highly efficient delivery, rapid intracellular release, and the accelerated signal output reaction, MnO2 nanosheets work better than commercial liposome carriers in live-cell biosensing analysis of survivin messenger RNA (mRNA), producing much brighter fluorescence images in a shorter time. The use of MnO2 nanosheets might provide a good carrier for different CRISPR/Cas systems and achieve the rapid and sensitive live-cell biosensing analysis of different intracellular targets, thus paving a promising way to promote the applications of CRISPR/Cas systems in living cells.}, }
@article {pmid35509363, year = {2022}, author = {Ebrahimi, S and Khanbabaei, H and Abbasi, S and Fani, M and Soltani, S and Zandi, M and Najafimemar, Z}, title = {CRISPR-Cas System: A Promising Diagnostic Tool for Covid-19.}, journal = {Avicenna journal of medical biotechnology}, volume = {14}, number = {1}, pages = {3-9}, pmid = {35509363}, issn = {2008-2835}, abstract = {More than a year has passed since the beginning of the 2019 novel coronavirus diseases (COVID-19) pandemic which has created massive problems globally affecting all aspects of people's life. Due to the emergence of new strains of the SARS-CoV-2, pandemic risk still remains, despite the start of vaccination. Therefore, rapid diagnostic tests are essential to control infection, improve clinical care and stop the spread of the disease. Recently CRISPR-based diagnostic tools have facilitated rapid diagnostic. Here, we review the diagnostic applications of CRISPR-Cas system in COVID-19.}, }
@article {pmid35508982, year = {2022}, author = {Shojaei Baghini, S and Gardanova, ZR and Abadi, SAH and Zaman, BA and İlhan, A and Shomali, N and Adili, A and Moghaddar, R and Yaseri, AF}, title = {CRISPR/Cas9 application in cancer therapy: a pioneering genome editing tool.}, journal = {Cellular &amp; molecular biology letters}, volume = {27}, number = {1}, pages = {35}, pmid = {35508982}, issn = {1689-1392}, mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Genome ; Humans ; *Neoplasms/genetics/therapy ; }, abstract = {The progress of genetic engineering in the 1970s brought about a paradigm shift in genome editing technology. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a flexible means to target and modify particular DNA sequences in the genome. Several applications of CRISPR/Cas9 are presently being studied in cancer biology and oncology to provide vigorous site-specific gene editing to enhance its biological and clinical uses. CRISPR's flexibility and ease of use have enabled the prompt achievement of almost any preferred alteration with greater efficiency and lower cost than preceding modalities. Also, CRISPR/Cas9 technology has recently been applied to improve the safety and efficacy of chimeric antigen receptor (CAR)-T cell therapies and defeat tumor cell resistance to conventional treatments such as chemotherapy and radiotherapy. The current review summarizes the application of CRISPR/Cas9 in cancer therapy. We also discuss the present obstacles and contemplate future possibilities in this context.}, }
@article {pmid35508977, year = {2022}, author = {Trung, NT and Son, LHP and Hien, TX and Quyen, DT and Bang, MH and Song, LH}, title = {CRISPR-Cas12a combination to alleviate the false-positive in loop-mediated isothermal amplification-based diagnosis of Neisseria meningitidis.}, journal = {BMC infectious diseases}, volume = {22}, number = {1}, pages = {429}, pmid = {35508977}, issn = {1471-2334}, support = {108.06-2017.21//Vietnam National Foundation for Science and Technology Development (NAFOSTED)/ ; 364/2020/HD-NCKHCN//The Vietnamese Ministry of National Defence/ ; }, mesh = {*CRISPR-Cas Systems ; DNA ; Humans ; Molecular Diagnostic Techniques ; *Neisseria meningitidis/genetics ; Nucleic Acid Amplification Techniques/methods ; RNA ; }, abstract = {BACKGROUND: Loop isothermal amplification (LAMP) has recently been proposed as a point-of-care diagnostic tool to detect acute infectious pathogens; however, this technique embeds risk of generating false-positive results. Whereas, with abilities to accurately recognize specific sequence, the CRISPR/Cas12a can forms complexes with cognate RNA sensors and cleave pathogen's DNA targets complimerntary to its cognate RNA, afterward acquiring the collateral activity to unbiasedly cut nearby off-target fragments. Therefore, if relevant fluorescent-quencher-nucleic probes are present in the reaction, the non-specific cleavage of probes releases fluorescences and establish diagnostic read-outs.<br><br>METHODS: The MetA gene of N. meningitidis was selected as target to optimize the LAMP reaction, whereas pseudo-dilution series of N. meningitidis gemonics DNA was used to establish the detection limit of LAMP/Cas12a combination assay. The diagnostic performance of established LAMP/Cas12a combination assay was validated in comparation with standard real-time PCR on 51 CSF samples (14 N. meningitidis confirmed patients and 37 control subjects).<br><br>RESULTS: In relevant biochemical conditions, CRISPR-Cas12a and LAMP can work synchronously to accurately identify genetics materials of Nesseria menitigistis at the level 40 copies/reaction less than 2 h.<br><br>CONCLUSIONS: In properly optimized conditions, the CRISPR-Cas12a system helps to alleviate false positive result hence enhancing the specificity of the LAMP assays.}, }
@article {pmid35508460, year = {2022}, author = {Mac Kain, A and Maarifi, G and Aicher, SM and Arhel, N and Baidaliuk, A and Munier, S and Donati, F and Vallet, T and Tran, QD and Hardy, A and Chazal, M and Porrot, F and OhAinle, M and Carlson-Stevermer, J and Oki, J and Holden, K and Zimmer, G and Simon-Lorière, E and Bruel, T and Schwartz, O and van der Werf, S and Jouvenet, N and Nisole, S and Vignuzzi, M and Roesch, F}, title = {Identification of DAXX as a restriction factor of SARS-CoV-2 through a CRISPR/Cas9 screen.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {2442}, pmid = {35508460}, issn = {2041-1723}, support = {ANR-20-COVI-000//Agence Nationale de la Recherche (French National Research Agency)/ ; }, mesh = {*COVID-19 ; CRISPR-Cas Systems ; Co-Repressor Proteins/genetics/metabolism ; Humans ; Interferons/metabolism ; Molecular Chaperones/genetics/metabolism ; Proteasome Endopeptidase Complex/metabolism ; *SARS-CoV-2 ; }, abstract = {Interferon restricts SARS-CoV-2 replication in cell culture, but only a handful of Interferon Stimulated Genes with antiviral activity against SARS-CoV-2 have been identified. Here, we describe a functional CRISPR/Cas9 screen aiming at identifying SARS-CoV-2 restriction factors. We identify DAXX, a scaffold protein residing in PML nuclear bodies known to limit the replication of DNA viruses and retroviruses, as a potent inhibitor of SARS-CoV-2 and SARS-CoV replication in human cells. Basal expression of DAXX is sufficient to limit the replication of SARS-CoV-2, and DAXX over-expression further restricts infection. DAXX restricts an early, post-entry step of the SARS-CoV-2 life cycle. DAXX-mediated restriction of SARS-CoV-2 is independent of the SUMOylation pathway but dependent on its D/E domain, also necessary for its protein-folding activity. SARS-CoV-2 infection triggers the re-localization of DAXX to cytoplasmic sites and promotes its degradation. Mechanistically, this process is mediated by the viral papain-like protease (PLpro) and the proteasome. Together, these results demonstrate that DAXX restricts SARS-CoV-2, which in turn has evolved a mechanism to counteract its action.}, }
@article {pmid35507186, year = {2022}, author = {Desjardins, J and Cowan, M and Yamanaka, Y}, title = {Designing Genetically Engineered Mouse Models (GEMMs) Using CRISPR Mediated Genome Editing.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2429}, number = {}, pages = {515-531}, pmid = {35507186}, issn = {1940-6029}, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Genome/genetics ; Mammals/genetics ; Mice ; RNA, Guide/genetics ; Zygote ; }, abstract = {Genetically engineered mouse models (GEMMs) are very powerful tools to study lineage hierarchy and cellular dynamics of stem cells in vivo. Stem cell behavior in various contexts such as development, normal homeostasis and diseases have been investigated using GEMMs. The strategies to generate GEMMs have drastically changed in the last decade with the development of the CRISPR/Cas9 system for manipulation of the mammalian genome. The advantages of the CRISPR/Cas9 are its simplicity and efficiency. The bioinformatics tools available now allow us to quickly identify appropriate guide RNAs and design experimental conditions to generate the targeted mutation. In addition, the genome can be manipulated directly in the zygote which reduces the time to modify target genes compared to other technologies such as Embryonic Stem (ES) cells. Equally important is that we can manipulate the genome of any mouse background with the CRISPR/Cas9 system which omits time-consuming backcrossing processes, accelerates research and increases flexibility. Here, we will summarize basic allelic types and our standard strategies of how to generate them.}, }
@article {pmid35507174, year = {2022}, author = {Ford, MJ and Yamanaka, Y}, title = {Reprogramming Mouse Oviduct Epithelial Cells Using In Vivo Electroporation and CRISPR/Cas9-Mediated Genetic Manipulation.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2429}, number = {}, pages = {367-377}, pmid = {35507174}, issn = {1940-6029}, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Electroporation/methods ; Epithelial Cells ; Fallopian Tubes ; Female ; *Gene Editing/methods ; Humans ; Mice ; }, abstract = {Advances in gene editing tools such as CRISPR/Cas9 have made precise in vivo gene editing possible, opening up avenues of research into somatic cell reprograming to study adult stem cells, homeostasis, and malignant transformation. Here we describe a method for CRISPR/Cas9 mediated in vivo gene editing, in combination with Cre-based lineage tracing via electroporation in the mouse oviduct. This method facilitates the delivery of multiple plasmids into oviduct epithelial cells, sufficient for studying homeostasis and generation of high-grade serous ovarian cancer (HGSOC) models.}, }
@article {pmid35507170, year = {2022}, author = {Devaraju, N and Rajendiran, V and Ravi, NS and Mohankumar, KM}, title = {Genome Engineering of Hematopoietic Stem Cells Using CRISPR/Cas9 System.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2429}, number = {}, pages = {307-331}, pmid = {35507170}, issn = {1940-6029}, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells ; Mice ; Transplantation, Autologous ; }, abstract = {Ex vivo genetic manipulation of autologous hematopoietic stem and progenitor cells (HSPCs) is a viable strategy for the treatment of hematologic and primary immune disorders. Targeted genome editing of HSPCs using the CRISPR-Cas9 system provides an effective platform to edit the desired genomic locus for therapeutic purposes with minimal off-target effects. In this chapter, we describe the detailed methodology for the CRISPR-Cas9 mediated gene knockout, deletion, addition, and correction in human HSPCs by viral and nonviral approaches. We also present a comprehensive protocol for the analysis of genome modified HSPCs toward the erythroid and megakaryocyte lineage in vitro and the long-term multilineage reconstitution capacity in the recently developed NBSGW mouse model that supports human erythropoiesis.}, }
@article {pmid35507169, year = {2022}, author = {Park, SH and Lee, CM and Bao, G}, title = {Identification and Validation of CRISPR/Cas9 Off-Target Activity in Hematopoietic Stem and Progenitor Cells.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2429}, number = {}, pages = {281-306}, pmid = {35507169}, issn = {1940-6029}, mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Hematopoietic Stem Cells/metabolism ; High-Throughput Nucleotide Sequencing ; *RNA, Guide/genetics/metabolism ; }, abstract = {Targeted genome editing in hematopoietic stem and progenitor cells (HSPCs) using CRISPR/Cas9 can potentially provide a permanent cure for hematologic diseases. However, the utility of CRISPR/Cas9 systems for therapeutic genome editing can be compromised by their off-target effects. In this chapter, we outline the procedures for CRISPR/Cas9 off-target identification and validation in HSPCs. This method is broadly applicable to diverse CRISPR/Cas9 systems and cell types. Using this protocol, researchers can perform computational prediction and experimental identification of potential off-target sites followed by off-target activity quantification by next-generation sequencing.}, }
@article {pmid35506667, year = {2022}, author = {Kever, L and Hardy, A and Luthe, T and Hünnefeld, M and Gätgens, C and Milke, L and Wiechert, J and Wittmann, J and Moraru, C and Marienhagen, J and Frunzke, J}, title = {Aminoglycoside Antibiotics Inhibit Phage Infection by Blocking an Early Step of the Infection Cycle.}, journal = {mBio}, volume = {}, number = {}, pages = {e0078322}, doi = {10.1128/mbio.00783-22}, pmid = {35506667}, issn = {2150-7511}, abstract = {In response to viral predation, bacteria have evolved a wide range of defense mechanisms, which rely mostly on proteins acting at the cellular level. Here, we show that aminoglycosides, a well-known class of antibiotics produced by Streptomyces, are potent inhibitors of phage infection in widely divergent bacterial hosts. We demonstrate that aminoglycosides block an early step of the viral life cycle, prior to genome replication. Phage inhibition was also achieved using supernatants from natural aminoglycoside producers, indicating a broad physiological significance of the antiviral properties of aminoglycosides. Strikingly, we show that acetylation of the aminoglycoside antibiotic apramycin abolishes its antibacterial effect but retains its antiviral properties. Altogether, our study expands the knowledge of aminoglycoside functions, suggesting that aminoglycosides not only are used by their producers as toxic molecules against their bacterial competitors but also could provide protection against the threat of phage predation at the community level. IMPORTANCE Predation by phages is a major driver of bacterial evolution. As a result, elucidating antiphage strategies is crucial from both fundamental and therapeutic standpoints. While protein-mediated defense mechanisms, like restriction-modification systems or CRISPR/Cas, have been extensively studied, much less is known about the potential antiphage activity of small molecules. Focusing on the model bacteria Escherichia coli and Streptomyces venezuelae, our findings revealed significant antiphage properties of aminoglycosides, a major class of translation-targeting antibiotics produced by Streptomyces. Further, we demonstrate that supernatants from natural aminoglycoside producers protect bacteria from phage propagation, highlighting the physiological relevance of this inhibition. Suppression of phage infection by aminoglycosides did not result from the indirect inhibition of bacterial translation, suggesting a direct interaction between aminoglycosides and phage components. This work highlights the molecular versatility of aminoglycosides, which have evolved to efficiently block protein synthesis in bacterial competitors and provide protection against phages.}, }
@article {pmid35506451, year = {2022}, author = {Kalafati, E and Papanikolaou, E and Marinos, E and Anagnou, NP and Pappa, KI}, title = {Mimiviruses: Giant viruses with novel and intriguing features (Review).}, journal = {Molecular medicine reports}, volume = {25}, number = {6}, pages = {}, doi = {10.3892/mmr.2022.12723}, pmid = {35506451}, issn = {1791-3004}, mesh = {*Amoeba ; CRISPR-Cas Systems ; Capsid ; *Giant Viruses/genetics ; Humans ; *Mimiviridae/genetics ; }, abstract = {The Mimivirus is a giant virus that infects amoebae and was long considered to be a bacterium due to its size. The viral particles are composed of a protein capsid of ~500 nm in diameter, which is enclosed in a polysaccharide layer in which ~120‑140 nm long fibers are embedded, resulting in an overall diameter of 700 nm. The virus has a genome size of 1.2 Mb DNA, and surprisingly, replicates only in the cytoplasm of the infected cells without entering the nucleus, which is a unique characteristic among DNA viruses. Their existence is undeniable; however, as with any novel discovery, there is still uncertainty concerning their pathogenicity mechanisms in humans and the nature of the Mimivirus virophage resistance element system (MIMIVIRE), a term given to describe the immune network of the Mimivirus, which closely resembles the CRISPR‑Cas system. The scope of the present review is to discuss the recent developments derived from structural and functional studies performed on the distinctive characteristics of the Mimivirus, and from studies concerning their putative clinical relevance in humans.}, }
@article {pmid35504953, year = {2022}, author = {Warmt, C and Yaslanmaz, C and Henkel, J}, title = {Investigation and validation of labelling loop mediated isothermal amplification (LAMP) products with different nucleotide modifications for various downstream analysis.}, journal = {Scientific reports}, volume = {12}, number = {1}, pages = {7137}, pmid = {35504953}, issn = {2045-2322}, mesh = {DNA Primers ; Molecular Diagnostic Techniques ; *Nucleic Acid Amplification Techniques/methods ; *Nucleotides ; Spectrometry, Fluorescence ; }, abstract = {Loop mediated isothermal amplification (LAMP) is one of the best known and most popular isothermal amplification methods. It's simplicity and speed make the method particularly suitable for point-of-care diagnostics. Nevertheless, false positive results remain a major drawback. Many (downstream) applications are known for the detection of LAMP amplicons like colorimetric assays, in-situ LAMP or CRISPR-Cas systems. Often, modifications of the LAMP products are necessary for different detection applications such as lateral flow assays. This is usually achieved with pre-modified primer. The aim of this study is to evaluate amplicon labelling with different modified nucleotides such as Cy5-dUTP, biotin-dUTP and aminoallyl-dUTP as an alternative to pre-labelled primers. To realise this, the effects on amplification and labelling efficiency were studied as a function of molecule size and nucleotide amount as well as target concentration. This research shows that diverse labelling of LAMP amplicons can be achieved using different, modified nucleotides during LAMP and that these samples can be analysed by a wide range of downstream applications such as fluorescence spectroscopy, gel electrophoresis, microarrays and lateral flow systems. Furthermore, microarray-based detection and the ability to identify and distinguish false positives were demonstrated as proof of concept.}, }
@article {pmid35504940, year = {2022}, author = {Sommerkamp, P and Sommerkamp, AC and Zeisberger, P and Eiben, PL and Narr, A and Korkmaz, A and Przybylla, A and Sohn, M and van der Hoeven, F and Schönig, K and Trumpp, A}, title = {CRISPR-Cas9 mediated generation of a conditional poly(A) binding protein nuclear 1 (Pabpn1) mouse model reveals an essential role for hematopoietic stem cells.}, journal = {Scientific reports}, volume = {12}, number = {1}, pages = {7181}, pmid = {35504940}, issn = {2045-2322}, support = {FOR2033//Deutsche Forschungsgemeinschaft/ ; RiskY-AML//Deutschen Konsortium für Translationale Krebsforschung/ ; SyTASC//Deutsche Krebshilfe/ ; }, mesh = {3' Untranslated Regions ; Animals ; *CRISPR-Cas Systems ; Disease Models, Animal ; Hematopoietic Stem Cells/metabolism ; Mice ; *Poly(A)-Binding Protein I/metabolism ; Polyadenylation ; RNA, Messenger/genetics ; }, abstract = {Poly(A) binding protein nuclear 1 (PABPN1) is known for its role in poly(A) tail addition and regulation of poly(A) tail length. In addition, it has been shown to be involved in alternative polyadenylation (APA). APA is a process regulating differential selection of polyadenylation sites, thereby influencing protein isoform expression and 3'-UTR make-up. In this study, we generated an inducible Pabpn1flox/flox mouse model using crRNA-tracrRNA:Cas9 complexes targeting upstream and downstream genomic regions, respectively, in combination with a long single-stranded DNA (ssDNA) template. We performed extensive in vitro testing of various guide RNAs (gRNAs) to optimize recombination efficiency for in vivo application. Pabpn1flox/flox mice were generated and crossed to MxCre mice for validation experiments, allowing the induction of Cre expression in the bone marrow (BM) by poly(I:C) (pIC) injections. Validation experiments revealed successful deletion of Pabpn1 and absence of PABPN1 protein. Functionally, knockout (KO) of Pabpn1 led to a rapid and robust depletion of hematopoietic stem and progenitor cells (HSPCs) as well as myeloid cells, suggesting an essential role of Pabpn1 in the hematopoietic lineage. Overall, the mouse model allows an inducible in-depth in vivo analysis of the role of PABPN1 and APA regulation in different tissues and disease settings.}, }
@article {pmid35502187, year = {2022}, author = {Espinoza-Mellado, MDR and Vilchis-Rangel, RE}, title = {Review of CRISPR-Cas Systems in Listeria Species: Current Knowledge and Perspectives.}, journal = {International journal of microbiology}, volume = {2022}, number = {}, pages = {9829770}, pmid = {35502187}, issn = {1687-918X}, abstract = {Listeria spp. are pathogens widely distributed in the environment and Listeria monocytogenes is associated with food-borne illness in humans. Food facilities represent an adverse environment for this bacterium, mainly due to the disinfection and cleaning processes included in good hygiene practices, and its virulence is related to stress responses. One of the recently described stress-response systems is CRISPR-Cas. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (cas) genes have been found in several bacteria. CRISPR-Cas has revolutionized biotechnology since it acts as an adaptive immune system of bacteria, which also helps in the evasion of the host immune response. There are three CRISPR systems described on Listeria species. Type II is present in many pathogenic bacteria and characterized by the presence of cas9 that becomes the main target of some anti-CRISPR proteins, such as AcrIIA1, encoded on Listeria phages. The presence of Cas9, either alone or in combination with anti-CRISPR proteins, suggests having a main role on the virulence of bacteria. In this review, we describe the most recent information on CRISPR-Cas systems in Listeria spp., particularly in L. monocytogenes, and their relationship with the virulence and pathogenicity of those bacteria. Besides, some applications of CRISPR systems and future challenges in the food processing industry, bacterial vaccination, antimicrobial resistance, pathogens biocontrol by phage therapy, and regulation of gene expression have been explored.}, }
@article {pmid35500195, year = {2022}, author = {Zhao, M and Gao, M and Xiong, L and Liu, Y and Tao, X and Gao, B and Liu, M and Wang, FQ and Wei, DZ}, title = {CRISPR-Cas Assisted Shotgun Mutagenesis Method for Evolutionary Genome Engineering.}, journal = {ACS synthetic biology}, volume = {}, number = {}, pages = {}, doi = {10.1021/acssynbio.2c00112}, pmid = {35500195}, issn = {2161-5063}, abstract = {Genome mutagenesis drives the evolution of organisms. Here, we developed a CRISPR-Cas assisted random mutation (CARM) technique for whole-genome mutagenesis. The method leverages an entirely random gRNA library and SpCas9-NG to randomly damage genomes in a controllable shotgunlike manner that then triggers diverse and abundant mutations via low-fidelity repair. As a proof of principle, CARM was applied to evolve the capacity of Saccharomyces cerevisiae BY4741 to produce β-carotene. After seven rounds of iterative evolution over two months, a β-carotene hyperproducing strain, C7-143, was isolated with a 10.5-fold increase in β-carotene production and 857 diverse genomic mutations that comprised indels, duplications, inversions, and chromosomal rearrangements. Transcriptomic analysis revealed that the expression of 2541 genes of strain C7-143 was significantly altered, suggesting that the metabolic landscape of the strain was deeply reconstructed. In addition, CARM was applied to evolve industrially relevant S. cerevisiae CEN.PK2-1C for S-adenosyl-L-methionine production, which was increased 2.28 times after just one round. Thus, CARM can contribute to increasing genetic diversity to identify new phenotypes that could further be investigated by reverse engineering.}, }
@article {pmid35499084, year = {2022}, author = {Caruso, SM and Quinn, PM and da Costa, BL and Tsang, SH}, title = {CRISPR/Cas therapeutic strategies for autosomal dominant disorders.}, journal = {The Journal of clinical investigation}, volume = {132}, number = {9}, pages = {}, pmid = {35499084}, issn = {1558-8238}, support = {R21 AG050437/AG/NIA NIH HHS/United States ; R01 EY018213/EY/NEI NIH HHS/United States ; U01 EY030580/EY/NEI NIH HHS/United States ; U54 OD020351/OD/NIH HHS/United States ; R01 EY009076/EY/NEI NIH HHS/United States ; R01 EY026682/EY/NEI NIH HHS/United States ; R24 EY027285/EY/NEI NIH HHS/United States ; P30 CA013696/CA/NCI NIH HHS/United States ; R24 EY028758/EY/NEI NIH HHS/United States ; R01 EY024698/EY/NEI NIH HHS/United States ; P30 EY019007/EY/NEI NIH HHS/United States ; }, mesh = {Alleles ; *CRISPR-Cas Systems ; Mutation ; }, abstract = {Autosomal dominant disorders present unique challenges, as therapeutics must often distinguish between healthy and diseased alleles while maintaining high efficiency, specificity, and safety. For this task, CRISPR/Cas remains particularly promising. Various CRISPR/Cas systems, like homology-directed repair, base editors, and prime editors, have been demonstrated to selectively edit mutant alleles either by incorporating these mutations into sgRNA sequences (near the protospacer-adjacent motif ["near the PAM"]) or by targeting a novel PAM generated by the mutation ("in the PAM"). However, these probability-based designs are not always assured, necessitating generalized, mutation-agnostic strategies like ablate-and-replace and single-nucleotide polymorphism editing. Here, we detail recent advancements in CRISPR therapeutics to treat a wide range of autosomal dominant disorders and discuss how they are altering the landscape for future therapies.}, }
@article {pmid35499048, year = {2022}, author = {Ibrahim, S and Saleem, B and Rehman, N and Zafar, SA and Naeem, MK and Khan, MR}, title = {CRISPR/Cas9 mediated disruption of Inositol Pentakisphosphate 2-Kinase 1 (TaIPK1) reduces phytic acid and improves iron and zinc accumulation in wheat grains.}, journal = {Journal of advanced research}, volume = {37}, number = {}, pages = {33-41}, pmid = {35499048}, issn = {2090-1224}, mesh = {CRISPR-Cas Systems ; Edible Grain ; Humans ; Inositol Phosphates ; Iron ; *Phytic Acid/metabolism ; *Triticum/genetics ; Zinc/metabolism ; }, abstract = {Introduction: Phytic acid (PA) is an important antinutrient agent present in cereal grains which reduces the bioavailability of iron and zinc in human body, causing malnutrition. Inositol pentakisphosphate 2- kinase 1 (IPK1) gene has been reported to be an important gene for PA biosynthesis.<br><br>Objective: A recent genome editing tool CRISPR/Cas9 has been successfully applied to develop biofortified rice by disrupting IPK1 gene, however, it remained a challenge in wheat. The aim of this study was to biofortify wheat using CRISPR/Cas9.<br><br>Methods: In this study, we isolated 3 TaIPK1 homeologs in wheat designated as TaIPK1.A, TaIPK1.B and TaIPK1.D and found that the expression abundance of TaIPK1.A was stronger in early stages of grain filling. Using CRISPR/Cas9, we have disrupted TaIPK1.A gene in cv. Borlaug-2016 with two guide RNAs targeting the 1st and 2nd exons.<br><br>Results: We got several genome-edited lines in the T0 generation at frequencies of 12.7% and 10.8%. Sequencing analysis revealed deletion of 1-23 nucleotides and even an addition of 1 nucleotide in various lines. Analysis of the genome-edited lines revealed a significant decrease in the PA content and an increase in iron and zinc accumulation in grains compared with control plants.<br><br>Conclusion: Our study demonstrates the potential application of CRISPR/Cas9 technique for the rapid generation of biofortified wheat cultivars.}, }
@article {pmid35496799, year = {2022}, author = {Picchi-Constante, GFA and Hiraiwa, PM and Marek, M and Rogerio, VZ and Guerra-Slompo, EP and Romier, C and Zanchin, NIT}, title = {Efficient CRISPR-Cas9-mediated genome editing for characterization of essential genes in Trypanosoma cruzi.}, journal = {STAR protocols}, volume = {3}, number = {2}, pages = {101324}, pmid = {35496799}, issn = {2666-1667}, mesh = {CRISPR-Cas Systems/genetics ; *Chagas Disease/genetics ; Gene Editing/methods ; Gene Knockout Techniques ; Genes, Essential ; Humans ; *Trypanosoma cruzi/genetics ; }, abstract = {This protocol outlines a new genetic complementation strategy to investigate gene function in Trypanosoma cruzi, the parasite causing Chagas disease. We combine CRISPR-Cas9 technology with recombination of variants of the target gene containing the desired mutations that are resistant to Cas9-cleavage, which enables detailed investigation of protein function. This experimental strategy overcomes some of the limitations associated with gene knockouts in T. cruzi. For complete details on the use and execution of this protocol, please refer to Marek et al. (2021).}, }
@article {pmid35496795, year = {2022}, author = {Ling, X and Chang, L and Chen, H and Liu, T}, title = {Efficient generation of locus-specific human CAR-T cells with CRISPR/cCas12a.}, journal = {STAR protocols}, volume = {3}, number = {2}, pages = {101321}, pmid = {35496795}, issn = {2666-1667}, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; }, abstract = {We recently developed a system to create human chimeric antigen receptor (CAR)-T cells using conjugated Cas12a (cCas12a) in which Cas12a is covalently linked to its CRISPR RNA (crRNA). This protocol describes site-specific modification of Cas12a and the preparation of Cas12a-crRNA complex using bio-orthogonal chemistry, followed by CAR-T cell generation through electroporation and AAV infection. This system shows robust editing efficiency in human cells and can be used for precisely targeted, highly efficient integration of CAR genes into T cell genome. For complete details on the use and execution of this protocol, please refer to Ling et al. (2021).}, }
@article {pmid35495695, year = {2022}, author = {Zhang, X and An, X}, title = {Adaptation by Type III CRISPR-Cas Systems: Breakthrough Findings and Open Questions.}, journal = {Frontiers in microbiology}, volume = {13}, number = {}, pages = {876174}, pmid = {35495695}, issn = {1664-302X}, abstract = {CRISPR-Cas systems acquire heritable defense memory against invading nucleic acids through adaptation. Type III CRISPR-Cas systems have unique and intriguing features of defense and are important in method development for Genetics research. We started to understand the common and unique properties of type III CRISPR-Cas adaptation in recent years. This review summarizes our knowledge regarding CRISPR-Cas adaptation with the emphasis on type III systems and discusses open questions for type III adaptation studies.}, }
@article {pmid35495653, year = {2022}, author = {Wörtz, J and Smith, V and Fallmann, J and König, S and Thuraisingam, T and Walther, P and Urlaub, H and Stadler, PF and Allers, T and Hille, F and Marchfelder, A}, title = {Cas1 and Fen1 Display Equivalent Functions During Archaeal DNA Repair.}, journal = {Frontiers in microbiology}, volume = {13}, number = {}, pages = {822304}, pmid = {35495653}, issn = {1664-302X}, abstract = {CRISPR-Cas constitutes an adaptive prokaryotic defence system against invasive nucleic acids like viruses and plasmids. Beyond their role in immunity, CRISPR-Cas systems have been shown to closely interact with components of cellular DNA repair pathways, either by regulating their expression or via direct protein-protein contact and enzymatic activity. The integrase Cas1 is usually involved in the adaptation phase of CRISPR-Cas immunity but an additional role in cellular DNA repair pathways has been proposed previously. Here, we analysed the capacity of an archaeal Cas1 from Haloferax volcanii to act upon DNA damage induced by oxidative stress and found that a deletion of the cas1 gene led to reduced survival rates following stress induction. In addition, our results indicate that Cas1 is directly involved in DNA repair as the enzymatically active site of the protein is crucial for growth under oxidative conditions. Based on biochemical assays, we propose a mechanism by which Cas1 plays a similar function to DNA repair protein Fen1 by cleaving branched intermediate structures. The present study broadens our understanding of the functional link between CRISPR-Cas immunity and DNA repair by demonstrating that Cas1 and Fen1 display equivalent roles during archaeal DNA damage repair.}, }
@article {pmid35491944, year = {2022}, author = {Fremin, BJ and Kyrpides, NC}, title = {Identifying candidate structured RNAs in CRISPR operons.}, journal = {RNA biology}, volume = {19}, number = {1}, pages = {678-685}, pmid = {35491944}, issn = {1555-8584}, mesh = {*CRISPR-Cas Systems ; Genomics ; Operon ; *RNA/genetics ; Repetitive Sequences, Nucleic Acid ; }, abstract = {Noncoding RNAs with secondary structures play important roles in CRISPR-Cas systems. Many of these structures likely remain undiscovered. We used a large-scale comparative genomics approach to predict 156 novel candidate structured RNAs from 36,111 CRISPR-Cas systems. A number of these were found to overlap with coding genes, including palindromic candidates that overlapped with a variety of Cas genes in type I and III systems. Among these 156 candidates, we identified 46 new models of CRISPR direct repeats and 1 tracrRNA. This tracrRNA model occasionally overlapped with predicted cas9 coding regions, emphasizing the importance of expanding our search windows for novel structure RNAs in coding regions. We also demonstrated that the antirepeat sequence in this tracrRNA model can be used to accurately assign thousands of predicted CRISPR arrays to type II-C systems. This study highlights the importance of unbiased identification of candidate structured RNAs across CRISPR-Cas systems.}, }
@article {pmid35490782, year = {2022}, author = {Tao, Y and Chaudhari, S and Shotorbani, PY and Ding, Y and Chen, Z and Kasetti, R and Zode, G and Ma, R}, title = {Enhanced Orai1-mediated store-operated Ca2+ channel/calpain signaling contributes to high glucose-induced podocyte injury.}, journal = {The Journal of biological chemistry}, volume = {}, number = {}, pages = {101990}, doi = {10.1016/j.jbc.2022.101990}, pmid = {35490782}, issn = {1083-351X}, abstract = {Podocyte injury induced by hyperglycemia is the main cause of kidney dysfunction in diabetic nephropathy. However, the underlying mechanism is unclear. Store-operated Ca2+ entry (SOCE) regulates a diversity of cellular processes in a variety of cell types. Calpain, a Ca2+-dependent cysteine protease, was recently shown to be involved in podocyte injury. In the present study, we sought to determine whether increased SOCE contributed to high glucose (HG)-induced podocyte injury through activation of the calpain pathway. In cultured human podocytes, whole-cell patch clamp indicated the presence of functional store-operated Ca2+ channels which are composed of Orai1 proteins and mediate SOCE. Western blots showed that HG treatment increased the protein abundance of Orai1 in a dose-dependent manner. Consistently, calcium imaging experiments revealed that SOCE was significantly enhanced in podocytes following HG treatment. Furthermore, HG treatment caused overt podocyte F-actin disorganization as well as a significant decrease in nephrin protein abundance, both of which are indications of podocyte injury. These podocyte injury responses were significantly blunted by both pharmacological inhibition of Orai1 using the small molecule inhibitor BTP2 or by genetic deletion of Orai1 using CRISPR-CAS9 lentivirus. Moreover, activation of SOCE by thapsigargin, an inhibitor of Ca2+ pump on the endoplasmic/sarcoplasmic reticulum membrane, significantly increased the activity of calpain, which was inhibited by BTP2. Finally, the calpain-1/-2 inhibitor calpeptin significantly blunted the nephrin protein reduction induced by HG treatment. Taken together, our results suggest that enhanced signaling via an Orai1/ SOCE/Calpain axis contributes to HG-induced podocyte injury.}, }
@article {pmid35487018, year = {2022}, author = {Klose, SM and Wawegama, N and Sansom, FM and Marenda, MS and Browning, GF}, title = {Efficient disruption of the function of the mnuA nuclease gene using the endogenous CRISPR/Cas system in Mycoplasma gallisepticum.}, journal = {Veterinary microbiology}, volume = {269}, number = {}, pages = {109436}, doi = {10.1016/j.vetmic.2022.109436}, pmid = {35487018}, issn = {1873-2542}, abstract = {Mycoplasmas are important animal pathogens, but the functions and roles of many of their genes in pathogenesis remain unclear, in large part because of the limited tools available for targeted mutagenesis in these bacteria. In this study we used the Mycoplasma gallisepticum CRISPR/Cas system to target a nuclease gene, MGA_0637 (mnuA), which is predicted to play a role in survival and virulence. Our strategy used simultaneous targeting of the ksgA kasugamycin resistance gene, as a mutation in this gene would not interfere with replication but would confer a readily detectable and selectable phenotype in transformants. A guide RNA plasmid, pKM-CRISPR, was constructed, with spacers targeting the ksgA and mnuA genes transcribed under the control of the vlhA1.1 promoter in a backbone plasmid carrying the oriC of M. imitans, and this plasmid was introduced into electrocompetent M. gallisepticum strain S6 cells. PCR assays targeting the ksgA gene, followed by Sanger sequence analyses of the phenotypically resistant transformants, detected polymorphisms within the targeted region of ksgA, confirming the activity of the endogenous CRISPR/Cas system. The nuclease activity of the kasugamycin resistant colonies was then assessed using zymogram assays. The complete or partial loss of nuclease activity in the majority of kasugamycin resistant isolates transformed with the CRISPR plasmid confirmed that the endogenous CRISPR/Cas system had effectively interfered with the function of both ksgA and mnuA genes. Sanger sequencing and RT-qPCR analyses of the mnuA gene suggested that the M. gallisepticum CRISPR/Cas system can be programmed to cleave both DNA and RNA.}, }
@article {pmid35483740, year = {2022}, author = {Herman, JA and Arora, S and Carter, L and Zhu, J and Biggins, S and Paddison, PJ}, title = {Functional dissection of human mitotic genes using CRISPR-Cas9 tiling screens.}, journal = {Genes &amp; development}, volume = {36}, number = {7-8}, pages = {495-510}, doi = {10.1101/gad.349319.121}, pmid = {35483740}, issn = {1549-5477}, support = {R01 GM064386/GM/NIGMS NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Mutagenesis ; }, abstract = {The identity of human protein-coding genes is well known, yet our in-depth knowledge of their molecular functions and domain architecture remains limited by shortcomings in homology-based predictions and experimental approaches focused on whole-gene depletion. To bridge this knowledge gap, we developed a method that leverages CRISPR-Cas9-induced mutations across protein-coding genes for the a priori identification of functional regions at the sequence level. As a test case, we applied this method to 48 human mitotic genes, revealing hundreds of regions required for cell proliferation, including domains that were experimentally characterized, ones that were predicted based on homology, and novel ones. We validated screen outcomes for 15 regions, including amino acids 387-402 of Mad1, which were previously uncharacterized but contribute to Mad1 kinetochore localization and chromosome segregation fidelity. Altogether, we demonstrate that CRISPR-Cas9-based tiling mutagenesis identifies key functional domains in protein-coding genes de novo, which elucidates separation of function mutants and allows functional annotation across the human proteome.}, }
@article {pmid35483732, year = {2022}, author = {Shin, J and Miller, M and Wang, YC}, title = {Recent advances in CRISPR-based systems for the detection of foodborne pathogens.}, journal = {Comprehensive reviews in food science and food safety}, volume = {}, number = {}, pages = {}, doi = {10.1111/1541-4337.12956}, pmid = {35483732}, issn = {1541-4337}, support = {ILLU-698-353//U.S. Department of Agriculture/ ; //University of Illinois Urbana-Champaign/ ; R20-7704//Royal Society of Chemistry/ ; }, abstract = {There has long been a need for more advanced forms of pathogen detection in the food industry. Though in its infancy, biosensing based on clustered regularly interspaced short palindromic repeats (CRISPR) has the potential to solve many problems that cannot be addressed using conventional methods. In this review, we briefly introduce and classify the various CRISPR/Cas protein effectors that have thus far been used in biosensors. We then assess the current state of CRISPR technology in food-safety contexts; describe how each Cas effector is utilized in foodborne-pathogen detection; and discuss the limitations of the current technology, as well as how it might usefully be applied in other areas of the food industry. We conclude that, if the limitations of existing CRISPR/Cas-based detection methods are overcome, they can be deployed on a wide scale and produce a range of positive food-safety outcomes.}, }
@article {pmid35481139, year = {2022}, author = {Li, Q and Feng, Q and Snouffer, A and Zhang, B and Rodríguez, GR and van der Knaap, E}, title = {Increasing Fruit Weight by Editing a Cis-Regulatory Element in Tomato KLUH Promoter Using CRISPR/Cas9.}, journal = {Frontiers in plant science}, volume = {13}, number = {}, pages = {879642}, pmid = {35481139}, issn = {1664-462X}, abstract = {CRISPR/Cas-mediated genome editing is a powerful approach to accelerate yield enhancement to feed growing populations. Most applications focus on "negative regulators" by targeting coding regions and promoters to create nulls or weak loss-of-function alleles. However, many agriculturally important traits are conferred by gain-of-function alleles. Therefore, creating gain-of-function alleles for "positive regulators" by CRISPR will be of great value for crop improvement. CYP78A family members are the positive regulators of organ weight and size in crops. In this study, we engineered allelic variation by editing tomato KLUH promoter around a single-nucleotide polymorphism (SNP) that is highly associated with fruit weight. The SNP was located in a conserved putative cis-regulatory element (CRE) as detected by the homology-based prediction and the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq). Twenty-one mutant alleles with various insertion and deletion sizes were generated in the LA1589 background. Five mutant alleles (m2+4bp , m3+1bp , m5-1bp , m13-8bp , and m14-9bp) showed a consistent increase in fruit weight and a significant decrease in the proportion of small fruits in all experimental evaluations. Notably, m2+4bp and m3+1bp homozygote significantly increase fruit weight by 10.7-15.7 and 8.7-16.3%, respectively. Further analysis of fruit weight based on fruit position on the inflorescence indicated that the five beneficial alleles increase the weight of all fruits along inflorescence. We also found that allele types and transcriptional changes of SlKLUH were poor predictors of the changes in fruit weight. This study not only provides a way of identifying conserved CRE but also highlights enormous potential for CRISPR/Cas-mediated cis-engineering of CYP78A members in yield improvement.}, }
@article {pmid35478255, year = {2022}, author = {Khairkhah, N and Bolhassani, A and Najafipour, R}, title = {Current and future direction in treatment of HPV-related cervical disease.}, journal = {Journal of molecular medicine (Berlin, Germany)}, volume = {}, number = {}, pages = {}, pmid = {35478255}, issn = {1432-1440}, abstract = {Human papillomavirus (HPV) is the most common sexually transmitted virus in the world. About 70% of cervical cancers are caused by the most oncogenic HPV genotypes of 16 and 18. Since available prophylactic vaccines do not induce immunity in those with established HPV infections, the development of therapeutic HPV vaccines using E6 and E7 oncogenes, or both as the target antigens remains essential. Also, knocking out the E6 and E7 oncogenes in host genome by genome-editing CRISPR/Cas system can result in tumor growth suppression. These methods have shown promising results in both preclinical and clinical trials and can be used for controlling the progression of HPV-related cervical diseases. This comprehensive review will detail the current treatment of HPV-related cervical precancerous and cancerous diseases. We also reviewed the future direction of treatment including different kinds of therapeutic methods and vaccines, genome-editing CRISPR/Cas system being studied in clinical trials. Although the progress in the development of therapeutic HPV vaccine has been slow, encouraging results from recent trials showed vaccine-induced regression in high-grade CIN lesions. CRISPR/Cas genome-editing system is also a promising strategy for HPV cancer therapy. However, its safety and specificity need to be optimized before it is used in clinical setting.}, }
@article {pmid35478023, year = {2022}, author = {Mallapaty, S}, title = {China focuses on ethics to deter another 'CRISPR babies' scandal.}, journal = {Nature}, volume = {605}, number = {7908}, pages = {15-16}, doi = {10.1038/d41586-022-01051-0}, pmid = {35478023}, issn = {1476-4687}, mesh = {*CRISPR-Cas Systems/genetics ; China ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Ethics, Medical ; *Gene Editing ; Humans ; }, }
@article {pmid35475170, year = {2022}, author = {Nemudraia, A and Nemudryi, A and Buyukyoruk, M and Scherffius, A and Zahl, T and Wiegand, T and Pandey, S and Nichols, J and Hall, L and McVey, A and Lee, H and Wilkinson, R and Snyder, L and Jones, J and Koutmou, K and Santiago-Frangos, A and Wiedenheft, B}, title = {Sequence-specific capture and concentration of viral RNA by type III CRISPR system enhances diagnostic.}, journal = {Research square}, volume = {}, number = {}, pages = {}, pmid = {35475170}, support = {R35 GM128836/GM/NIGMS NIH HHS/United States ; R35 GM134867/GM/NIGMS NIH HHS/United States ; }, abstract = {Type-III CRISPR-Cas systems have recently been adopted for sequence-specific detection of SARS-CoV-2. Here, we make two major advances that simultaneously limit sample handling and significantly enhance the sensitivity of SARS-CoV-2 RNA detection directly from patient samples. First, we repurpose the type III-A CRISPR complex from Thermus thermophilus (TtCsm) for programmable capture and concentration of specific RNAs from complex mixtures. The target bound TtCsm complex primarily generates two cyclic oligoadenylates (i.e., cA3 and cA4) that allosterically activate ancillary nucleases. To improve sensitivity of the diagnostic, we identify and test several ancillary nucleases (i.e., Can1, Can2, and NucC). We show that Can1 and Can2 are activated by both cA3 and cA4, and that different activators trigger changes in the substrate specificity of these nucleases. Finally, we integrate the type III-A CRISPR RNA-guided capture technique with the Can2 nuclease for 90 fM (5x104 copies/ul) detection of SARS-CoV-2 RNA directly from nasopharyngeal swab samples.}, }
@article {pmid35474904, year = {2022}, author = {Cheng, L and Yang, F and Tang, L and Qian, L and Chen, X and Guan, F and Zhang, J and Li, G}, title = {Electrochemical Evaluation of Tumor Development via Cellular Interface Supported CRISPR/Cas Trans-Cleavage.}, journal = {Research (Washington, D.C.)}, volume = {2022}, number = {}, pages = {9826484}, pmid = {35474904}, issn = {2639-5274}, abstract = {Evaluating tumor development is of great importance for clinic treatment and therapy. It has been known that the amounts of sialic acids on tumor cell membrane surface are closely associated with the degree of cancerization of the cell. So, in this work, cellular interface supported CRISPR/Cas trans-cleavage has been explored for electrochemical simultaneous detection of two types of sialic acids, i.e., N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac). Specifically, PbS quantum dot-labeled DNA modified by Neu5Gc antibody is prepared to specifically recognize Neu5Gc on the cell surface, followed by the binding of Neu5Ac through our fabricated CdS quantum dot-labeled DNA modified by Sambucus nigra agglutinin. Subsequently, the activated Cas12a indiscriminately cleaves DNA, resulting in the release of PbS and CdS quantum dots, both of which can be simultaneously detected by anodic stripping voltammetry. Consequently, Neu5Gc and Neu5Ac on cell surface can be quantitatively analyzed with the lowest detection limits of 1.12 cells/mL and 1.25 cells/mL, respectively. Therefore, a ratiometric electrochemical method can be constructed for kinetic study of the expression and hydrolysis of Neu5Gc and Neu5Ac on cell surface, which can be further used as a tool to identify bladder cancer cells at different development stages. Our method to evaluate tumor development is simple and easy to be operated, so it can be potentially applied for the detection of tumor occurrence and development in the future.}, }
@article {pmid35473305, year = {2022}, author = {Bindal, G and Amlinger, L and Lundgren, M and Rath, D}, title = {Type I-E CRISPR-Cas System as a Defense System in Saccharomyces cerevisiae.}, journal = {mSphere}, volume = {}, number = {}, pages = {e0003822}, doi = {10.1128/msphere.00038-22}, pmid = {35473305}, issn = {2379-5042}, abstract = {Defense against viruses and other mobile genetic elements (MGEs) is important in many organisms. The CRISPR-Cas systems found in bacteria and archaea constitute adaptive immune systems that can acquire the ability to target previously unrecognized MGEs. No CRISPR-Cas system is found to occur naturally in eukaryotic cells, but here, we demonstrate interference by a type I-E CRISPR-Cas system from Escherichia coli introduced in Saccharomyces cerevisiae. The designed CRISPR arrays are expressed and processed properly in S. cerevisiae. Targeted plasmids display reduced transformation efficiency, indicative of DNA cleavage. IMPORTANCE Genetic inactivation of viruses and other MGEs is an important tool with application in both research and therapy. Gene editing using, e.g., Cas9-based systems, can be used to inactivate MGEs in eukaryotes by introducing specific mutations. However, type I-E systems processively degrade the target which allows for inactivation without detailed knowledge of gene function. A reconstituted CRISPR-Cas system in S. cerevisiae can also function as a basic research platform for testing the role of various factors in the interference process.}, }
@article {pmid35472302, year = {2022}, author = {Cho, SI and Lee, S and Mok, YG and Lim, K and Lee, J and Lee, JM and Chung, E and Kim, JS}, title = {Targeted A-to-G base editing in human mitochondrial DNA with programmable deaminases.}, journal = {Cell}, volume = {185}, number = {10}, pages = {1764-1776.e12}, doi = {10.1016/j.cell.2022.03.039}, pmid = {35472302}, issn = {1097-4172}, mesh = {Animals ; CRISPR-Cas Systems ; Cytosine/metabolism ; *DNA, Mitochondrial/genetics ; Escherichia coli/genetics/metabolism ; Gene Editing ; Humans ; Mitochondria/genetics/metabolism ; *Mitochondrial Diseases/genetics ; Purines ; }, abstract = {Mitochondrial DNA (mtDNA) editing paves the way for disease modeling of mitochondrial genetic disorders in cell lines and animals and also for the treatment of these diseases in the future. Bacterial cytidine deaminase DddA-derived cytosine base editors (DdCBEs) enabling mtDNA editing, however, are largely limited to C-to-T conversions in the 5'-TC context (e.g., TC-to-TT conversions), suitable for generating merely 1/8 of all possible transition (purine-to-purine and pyrimidine-to-pyrimidine) mutations. Here, we present transcription-activator-like effector (TALE)-linked deaminases (TALEDs), composed of custom-designed TALE DNA-binding arrays, a catalytically impaired, full-length DddA variant or split DddA originated from Burkholderia cenocepacia, and an engineered deoxyadenosine deaminase derived from the E. coli TadA protein, which induce targeted A-to-G editing in human mitochondria. Custom-designed TALEDs were highly efficient in human cells, catalyzing A-to-G conversions at a total of 17 target sites in various mitochondrial genes with editing frequencies of up to 49%.}, }
@article {pmid35472287, year = {2022}, author = {Horáčková, V and Voleman, L and Hagen, KD and Petrů, M and Vinopalová, M and Weisz, F and Janowicz, N and Marková, L and Motyčková, A and Najdrová, V and Tůmová, P and Dawson, SC and Doležal, P}, title = {Efficient CRISPR/Cas9-mediated gene disruption in the tetraploid protist Giardia intestinalis.}, journal = {Open biology}, volume = {12}, number = {4}, pages = {210361}, pmid = {35472287}, issn = {2046-2441}, mesh = {*CRISPR-Cas Systems ; Gene Editing/methods ; *Giardia lamblia/genetics ; Humans ; RNA, Guide ; Tetraploidy ; }, abstract = {CRISPR/Cas9-mediated genome editing has become an extremely powerful technique used to modify gene expression in many organisms, including parasitic protists. Giardia intestinalis, a protist parasite that infects approximately 280 million people around the world each year, has been eluding the use of CRISPR/Cas9 to generate knockout cell lines due to its tetraploid genome. In this work, we show the ability of the in vitro assembled CRISPR/Cas9 components to successfully edit the genome of G. intestinalis. The cell line that stably expresses Cas9 in both nuclei of G. intestinalis showed effective recombination of the cassette containing the transcription units for the gRNA and the resistance marker. This highly efficient process led to the removal of all gene copies at once for three independent experimental genes, mem, cwp1 and mlf1. The method was also applicable to incomplete disruption of the essential gene, as evidenced by significantly reduced expression of tom40. Finally, testing the efficiency of Cas9-induced recombination revealed that homologous arms as short as 150 bp can be sufficient to establish a complete knockout cell line in G. intestinalis.}, }
@article {pmid35470620, year = {2022}, author = {LE, Y and He, X and Sun, J}, title = {[Thermostable CRISPR/Cas9 genome editing system and its application in construction of cell factories with thermophilic bacteria: a review].}, journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology}, volume = {38}, number = {4}, pages = {1475-1489}, doi = {10.13345/j.cjb.210827}, pmid = {35470620}, issn = {1872-2075}, mesh = {Bacteria/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Genome ; }, abstract = {The diverse thermophilic strains of Thermoanaerobacter, serving as unique platforms with a broad range of application in biofuels and chemicals, have received wide attention from scholars and practitioners. Although biochemical experiments and genome sequences have been reported for a variety of Thermoanaerobacter strains, an efficient genetic manipulation system remains to be established for revealing the biosynthetic pathways of Thermoanaerobacter. In line with this demand, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) systems for editing, regulating and targeting genomes have been well developed in thermophiles. Here, we reviewed and discussed the current status, associated challenges, and future perspectives of the construction of thermostable CRISPR/Cas9 genome editing systems for some representative Thermoanaerobacter species. The establishment, optimization, and application of thermostable CRISPR/Cas genome editing systems would potentially provide a foundation for further genetic modification of thermophilic bacteria.}, }
@article {pmid35470618, year = {2022}, author = {Chao, S and Hu, X}, title = {[Application of gene editing technology in Escherichia coli].}, journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology}, volume = {38}, number = {4}, pages = {1446-1461}, doi = {10.13345/j.cjb.210680}, pmid = {35470618}, issn = {1872-2075}, mesh = {CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; *Gene Editing ; Homologous Recombination ; Technology ; }, abstract = {Gene editing technology can be used to modify the genome of Escherichia coli for the investigation of gene functions, or to change the metabolic pathways for the efficient production of high-value products in engineered strains with genetic stability. A variety of gene editing technologies have been applied in prokaryotes, such as λ-Red homologous recombination and CRISPR/Cas9. As a traditional gene editing technique, λ-Red recombination is widely used. However, it has a few shortcomings, such as the limited integration efficiency by the integrated fragment size, the cumbersome gene editing process, and the FRT scar in the genome after recombination. CRISPR/Cas9 is widely used for genome editing at specific sites, which requires specific DNA segments according to the editing site. As the understanding of the two technologies deepens, a variety of composite gene editing techniques have been developed, such as the application of λ-Red homologous recombination in combination with homing endonucleaseⅠ-SceⅠ or CRISPR/Cas9. In this review, we summarized the basic principles of common gene editing techniques and composite gene editing techniques, as well as their applications in Escherichia coli, which can provide a basis for the selection of gene editing methods in prokaryotes.}, }
@article {pmid35470617, year = {2022}, author = {Wang, C and Liu, Z and Tang, B and Yang, H and Sun, D}, title = {[Prevention and control of antimicrobial resistance using CRISPR-Cas system: a review].}, journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology}, volume = {38}, number = {4}, pages = {1432-1445}, doi = {10.13345/j.cjb.210348}, pmid = {35470617}, issn = {1872-2075}, mesh = {Anti-Bacterial Agents ; *Bacteriophages/genetics ; *CRISPR-Cas Systems ; Drug Resistance, Bacterial/genetics ; Plasmids/genetics ; }, abstract = {Bacterial multi-drug resistance (MDR) is a global challenge in the fields of medicine and health, agriculture and fishery, ecology and environment. The cross-region spread of antibiotic resistance genes (ARGs) among different species is one of the main cause of bacterial MDR. However, there is no effective strategies for addressing the intensifying bacterial MDR. The CRISPR-Cas system, consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated proteins, can targetedly degrade exogenous nucleic acids, thus exhibiting high application potential in preventing and controlling bacterial MDR caused by ARGs. This review briefly introduced the working mechanism of CRISPR-Cas systems, followed by discussing recent advances in reducing ARGs by CRISPR-Cas systems delivered through mediators (e.g. plasmids, bacteriophages and nanoparticle). Moreover, the trends of this research field were envisioned, providing a new perspective on preventing and controlling MDR.}, }
@article {pmid35469710, year = {2022}, author = {Cury, J and Bernheim, A}, title = {CRISPR-Cas and restriction-modification team up to achieve long-term immunity.}, journal = {Trends in microbiology}, volume = {30}, number = {6}, pages = {513-514}, doi = {10.1016/j.tim.2022.04.001}, pmid = {35469710}, issn = {1878-4380}, mesh = {Bacteria/genetics ; *Bacteriophages/genetics ; *CRISPR-Cas Systems ; }, abstract = {Bacteria have been shown to harbor a growing arsenal of various defense systems against phages. Maguin et al. have uncovered how two of the most frequent defense systems interact: the clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) system recycles by-products of the restriction-modification (RM) system to increase bacterial defense in the long run.}, }
@article {pmid35468950, year = {2022}, author = {Mitkas, AA and Valverde, M and Chen, W}, title = {Dynamic modulation of enzyme activity by synthetic CRISPR-Cas6 endonucleases.}, journal = {Nature chemical biology}, volume = {18}, number = {5}, pages = {492-500}, pmid = {35468950}, issn = {1552-4469}, support = {CBET1803008, MCB1817675 and MCB2013991//National Science Foundation (NSF)/ ; }, mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Endonucleases/metabolism ; RNA/metabolism ; RNA Processing, Post-Transcriptional ; }, abstract = {In nature, dynamic interactions between enzymes play a crucial role in defining cellular metabolism. By controlling the spatial and temporal organization of these supramolecular complexes called metabolons, natural metabolism can be tuned in a highly dynamic manner. Here, we repurpose the CRISPR-Cas6 family proteins as a synthetic strategy to create dynamic metabolons by combining the ease of RNA processing and the predictability of RNA hybridization for protein assembly. By disturbing RNA-RNA networks using toehold-mediated strand displacement reactions, on-demand assembly and disassembly are achieved using both synthetic RNA triggers and mCherry messenger RNA. Both direct and 'Turn-On' assembly of the pathway enzymes tryptophan-2-monooxygenase and indoleacetamide hydrolase can enhance indole-3-acetic acid production by up to ninefold. Even multimeric enzymes can be assembled to improve malate production by threefold. By interfacing with endogenous mRNAs, more complex metabolons may be constructed, resulting in a self-responsive metabolic machinery capable of adapting to changing cellular demand.}, }
@article {pmid35468907, year = {2022}, author = {Thean, DGL and Chu, HY and Fong, JHC and Chan, BKC and Zhou, P and Kwok, CCS and Chan, YM and Mak, SYL and Choi, GCG and Ho, JWK and Zheng, Z and Wong, ASL}, title = {Machine learning-coupled combinatorial mutagenesis enables resource-efficient engineering of CRISPR-Cas9 genome editor activities.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {2219}, pmid = {35468907}, issn = {2041-1723}, support = {32022089//National Natural Science Foundation of China (National Science Foundation of China)/ ; 17104619//Research Grants Council, University Grants Committee (RGC, UGC)/ ; }, mesh = {*Bacterial Proteins/metabolism ; *CRISPR-Cas Systems/genetics ; DNA/metabolism ; Humans ; Machine Learning ; Mutagenesis ; }, abstract = {The genome-editing Cas9 protein uses multiple amino-acid residues to bind the target DNA. Considering only the residues in proximity to the target DNA as potential sites to optimise Cas9's activity, the number of combinatorial variants to screen through is too massive for a wet-lab experiment. Here we generate and cross-validate ten in silico and experimental datasets of multi-domain combinatorial mutagenesis libraries for Cas9 engineering, and demonstrate that a machine learning-coupled engineering approach reduces the experimental screening burden by as high as 95% while enriching top-performing variants by ∼7.5-fold in comparison to the null model. Using this approach and followed by structure-guided engineering, we identify the N888R/A889Q variant conferring increased editing activity on the protospacer adjacent motif-relaxed KKH variant of Cas9 nuclease from Staphylococcus aureus (KKH-SaCas9) and its derived base editor in human cells. Our work validates a readily applicable workflow to enable resource-efficient high-throughput engineering of genome editor's activity.}, }
@article {pmid35468792, year = {2022}, author = {Huang, H and Huang, G and Tan, Z and Hu, Y and Shan, L and Zhou, J and Zhang, X and Ma, S and Lv, W and Huang, T and Liu, Y and Wang, D and Zhao, X and Lin, Y and Rong, Z}, title = {Engineered Cas12a-Plus nuclease enables gene editing with enhanced activity and specificity.}, journal = {BMC biology}, volume = {20}, number = {1}, pages = {91}, pmid = {35468792}, issn = {1741-7007}, support = {2017YFA0105001//the National Key R&amp;D Program of China/ ; 82070002, 81872511//the National Natural Science Foundation of China/ ; 82072329//the National Natural Science Foundation of China/ ; 2021M701615//the Fellowship of China Postdoctoral Science Foundation/ ; 2021M691473//the Fellowship of China Postdoctoral Science Foundation/ ; 2021M701634//the Fellowship of China Postdoctoral Science Foundation/ ; }, mesh = {Acidaminococcus/genetics ; *CRISPR-Cas Systems ; Endonucleases/genetics ; *Gene Editing ; Proto-Oncogene Proteins B-raf/genetics ; }, abstract = {BACKGROUND: The CRISPR-Cas12a (formerly Cpf1) system is a versatile gene-editing tool with properties distinct from the broadly used Cas9 system. Features such as recognition of T-rich protospacer-adjacent motif (PAM) and generation of sticky breaks, as well as amenability for multiplex editing in a single crRNA and lower off-target nuclease activity, broaden the targeting scope of available tools and enable more accurate genome editing. However, the widespread use of the nuclease for gene editing, especially in clinical applications, is hindered by insufficient activity and specificity despite previous efforts to improve the system. Currently reported Cas12a variants achieve high activity with a compromise of specificity. Here, we used structure-guided protein engineering to improve both editing efficiency and targeting accuracy of Acidaminococcus sp. Cas12a (AsCas12a) and Lachnospiraceae bacterium Cas12a (LbCas12a).<br><br>RESULTS: We created new AsCas12a variant termed "AsCas12a-Plus" with increased activity (1.5~2.0-fold improvement) and specificity (reducing off-targets from 29 to 23 and specificity index increased from 92% to 94% with 33 sgRNAs), and this property was retained in multiplex editing and transcriptional activation. When used to disrupt the oncogenic BRAFV600E mutant, AsCas12a-Plus showed less off-target activity while maintaining comparable editing efficiency and BRAFV600E cancer cell killing. By introducing the corresponding substitutions into LbCas12a, we also generated LbCas12a-Plus (activity improved ~1.1-fold and off-targets decreased from 20 to 12 while specificity index increased from 78% to 89% with 15 sgRNAs), suggesting this strategy may be generally applicable across Cas12a orthologs. We compared Cas12a-Plus, other variants described in this study, and the reported enCas12a-HF, enCas12a, and Cas12a-ultra, and found that Cas12a-Plus outperformed other variants with a good balance for enhanced activity and improved specificity.<br><br>CONCLUSIONS: Our discoveries provide alternative AsCas12a and LbCas12a variants with high specificity and activity, which expand the gene-editing toolbox and can be more suitable for clinical applications.}, }
@article {pmid35467081, year = {2022}, author = {Sparmann, A and Beisel, CL}, title = {CRISPR memories in single cells.}, journal = {Molecular systems biology}, volume = {18}, number = {4}, pages = {e11011}, pmid = {35467081}, issn = {1744-4292}, mesh = {*Bacteria/genetics ; *CRISPR-Cas Systems ; }, abstract = {CRISPR-Cas systems allow bacteria to memorize prior infections as a means to combat the same invader if it attempts another attack in the future. While the underlying mechanisms of this bacterial immunity have been intensely studied over the past decade, little attention has been paid to CRISPR defense at the single-cell level. In their recent work, Brouns and colleagues (McKenzie et al, 2022) track memory acquisition and defense in individual cells and find a wide range of temporal dynamics that shape how a cell population experiences and combats an active infection.}, }
@article {pmid35467080, year = {2022}, author = {McKenzie, RE and Keizer, EM and Vink, JNA and van Lopik, J and Büke, F and Kalkman, V and Fleck, C and Tans, SJ and Brouns, SJJ}, title = {Single cell variability of CRISPR-Cas interference and adaptation.}, journal = {Molecular systems biology}, volume = {18}, number = {4}, pages = {e10680}, doi = {10.15252/msb.202110680}, pmid = {35467080}, issn = {1744-4292}, support = {801041//European Union Horizons 2020, CyGenTiG/ ; 101003229//EC|H2020|H2020 Priority Excellent Science|H2020 European Research Council (ERC)/ ; //NWO Frontiers of Nanoscience Nanofront/ ; VI.C.182.027//Netherlands Organisation for Scientific Research NWO vici/ ; }, mesh = {Adaptation, Physiological/genetics ; *Bacteriophages ; *CRISPR-Cas Systems/genetics ; DNA/metabolism ; Escherichia coli/genetics/metabolism ; }, abstract = {While CRISPR-Cas defence mechanisms have been studied on a population level, their temporal dynamics and variability in individual cells have remained unknown. Using a microfluidic device, time-lapse microscopy and mathematical modelling, we studied invader clearance in Escherichia coli across multiple generations. We observed that CRISPR interference is fast with a narrow distribution of clearance times. In contrast, for invaders with escaping PAM mutations we found large cell-to-cell variability, which originates from primed CRISPR adaptation. Faster growth and cell division and higher levels of Cascade increase the chance of clearance by interference, while slower growth is associated with increased chances of clearance by priming. Our findings suggest that Cascade binding to the mutated invader DNA, rather than spacer integration, is the main source of priming heterogeneity. The highly stochastic nature of primed CRISPR adaptation implies that only subpopulations of bacteria are able to respond quickly to invading threats. We conjecture that CRISPR-Cas dynamics and heterogeneity at the cellular level are crucial to understanding the strategy of bacteria in their competition with other species and phages.}, }
@article {pmid35466160, year = {2022}, author = {Bajaj, A and Cuchel, M}, title = {Advancements in the Treatment of Homozygous Familial Hypercholesterolemia.}, journal = {Journal of atherosclerosis and thrombosis}, volume = {}, number = {}, pages = {}, doi = {10.5551/jat.RV17065}, pmid = {35466160}, issn = {1880-3873}, abstract = {Homozygous familial hypercholesterolemia (HoFH) is a rare genetic disorder with extreme elevations of low-density lipoprotein cholesterol (LDL-C) leading to premature atherosclerotic cardiovascular disease (ASCVD) as early as in childhood. Management of HoFH centers around aggressive and adequate reduction of LDL-C levels to slow the trajectory of ASCVD development. Historically, lowering LDL-C levels in HoFH has been challenging because of both the markedly elevated LDL-C levels (often ＞400 mg/dL) and reduced response to treatment options, such as statins, for which the mechanism of action requires a functional LDL receptor. However, the treatment landscape for HoFH has rapidly progressed over the last decade. While statins and ezetimibe remain first-line treatment, patients often require addition of multiple therapies to achieve goal LDL-C levels. The PCSK9 inhibitors are an important recent addition to the available treatment options, along with lomitapide, bile acid sequestrants, and, possibly, bempedoic acid. Additionally, ANGPTL3 has emerged as an important therapeutic target, with evinacumab being the first available ANGPTL3 inhibitor on the market for the treatment of patients with HoFH. For patients who cannot achieve adequate LDL-C reduction, lipoprotein apheresis may be necessary, with the added benefit of reducing lipoprotein(a) levels that carries an added risk if also elevated in patients with HoFH. Finally, gene therapy and genome editing using CRISPR/Cas-9 are moving through clinical development and may dramatically alter the future landscape of treatment for HoFH.}, }
@article {pmid35465826, year = {2022}, author = {Ghosh, A and Myacheva, K and Riester, M and Schmidt, C and Diederichs, S}, title = {Chimeric oligonucleotides combining guide RNA and single-stranded DNA repair template effectively induce precision gene editing.}, journal = {RNA biology}, volume = {19}, number = {1}, pages = {588-593}, doi = {10.1080/15476286.2022.2067713}, pmid = {35465826}, issn = {1555-8584}, mesh = {CRISPR-Cas Systems ; Chimera/metabolism ; DNA, Single-Stranded/genetics ; *Gene Editing/methods ; Oligonucleotides/genetics ; *RNA, Guide/genetics ; Ribonucleoproteins/metabolism ; }, abstract = {The ability to precisely alter the genome holds immense potential for molecular biology, medicine and biotechnology. The development of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) into a genomic editing tool has vastly simplified genome engineering. Here, we explored the use of chemically synthesized chimeric oligonucleotides encoding a target-specific crRNA (CRISPR RNA) fused to a single-stranded DNA repair template for RNP-mediated precision genome editing. By generating three clinically relevant oncogenic driver mutations, two non-stop extension mutations, an FGFRi resistance mutation and a single nucleotide change, we demonstrate the ability of chimeric oligos to form RNPs and direct Cas9 to effectively induce genome editing. Further, we demonstrate that the polarity of the chimeric oligos is crucial: only chimeric oligos with the single-stranded DNA repair template fused to the 3'-end of the crRNA are functional for accurate editing, while templates fused to the 5'-end are ineffective. We also find that chimeras can perform editing with both symmetric and asymmetric single-stranded DNA repair templates. Depending on the target locus, the editing efficiency using chimeric RNPs is similar to or less than the efficiency of editing using the bipartite standard RNPs. Our results indicate that chimeric RNPs comprising RNA-DNA oligos formed from fusing the crRNA and DNA repair templates can successfully induce precise edits. While chimeric RNPs do not display an advantage over standard RNPs, they nonetheless represent a viable approach for one-molecule precision genome editing.}, }
@article {pmid35461863, year = {2022}, author = {Yadav, M and Atala, A and Lu, B}, title = {Developing all-in-one virus-like particles for Cas9 mRNA/single guide RNA co-delivery and aptamer-containing lentiviral vectors for improved gene expression.}, journal = {International journal of biological macromolecules}, volume = {209}, number = {Pt A}, pages = {1260-1270}, doi = {10.1016/j.ijbiomac.2022.04.114}, pmid = {35461863}, issn = {1879-0003}, mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Gene Expression ; Gene Products, gag/genetics ; Oligonucleotides ; *RNA, Guide/genetics ; RNA, Messenger/genetics ; }, abstract = {Lentiviral vectors (LVs) are widely used for delivering foreign genes for long-term expression. Recently, virus-like particles (VLPs) were developed for mRNA or ribonucleoprotein (RNP) delivery for short-term endonuclease expression. Generating large amount of LVs or VLPs is challenging. On the other hand, methods for using VLPs to co-deliver Cas9 mRNA and single guide RNA (sgRNA) are limited. Fusing aptamer-binding protein (ABP) to the N-terminus of HIV Gag protein is currently the successful way to develop hybrid particles for co-delivering Cas9 mRNA and sgRNA. The effects of modifying Gag protein this way on particle assembly are unknown. Previously we found that adding an ABP after the second zinc finger domain of nucleocapsid (NC) protein had minimal effects on particle assembly. Based on these observations, here we developed hybrid particles for Cas9 mRNA and sgRNA co-delivery with normal capsid assembly efficiency. We further improved LVs for integrated gene expression by including an aptamer sequence in lentiviral genomic RNA, which improved lentiviral particle production and enhanced LV genomic RNA packaging. In summary, here we describe the development of new all-in-one VLPs for co-delivery of Cas9 mRNA and sgRNA, and new LVs for enhanced vector production and gene expression.}, }
@article {pmid35461662, year = {2022}, author = {Le, Y and Sun, J}, title = {CRISPR/Cas genome editing systems in thermophiles: Current status, associated challenges, and future perspectives.}, journal = {Advances in applied microbiology}, volume = {118}, number = {}, pages = {1-30}, doi = {10.1016/bs.aambs.2022.02.001}, pmid = {35461662}, issn = {0065-2164}, mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; *Gene Editing ; Metabolic Engineering ; }, abstract = {Thermophiles, offering an attractive and unique platform for a broad range of applications in biofuels and environment protections, have received a significant attention and growing interest from academy and industry. However, the exploration and exploitation of thermophilic organisms have been hampered by the lack of a powerful genome manipulation tool to improve production efficiency. At current, the clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR associated (Cas) system has been successfully exploited as a competent, simplistic, and powerful tool for genome engineering both in eukaryotes and prokaryotes. Indeed, with the significant efforts made in recent years, some thermostable Cas9 proteins have been well identified and characterized and further, some thermostable Cas9-based editing tools have been successfully established in some representative obligate thermophiles. In this regard, we reviewed the current status and its progress in CRISPR/Cas-based genome editing system towards a variety of thermophilic organisms. Despite the potentials of these progresses, multiple factors/barriers still have to be overcome and optimized for improving its editing efficiency in thermophiles. Some insights into the roles of thermostable CRISPR/Cas technologies for the metabolic engineering of thermophiles as a thermophilic microbial cell factory were also fully analyzed and discussed.}, }
@article {pmid35461452, year = {2022}, author = {Bieluszewski, T and Szymanska-Lejman, M and Dziegielewski, W and Zhu, L and Ziolkowski, PA}, title = {Efficient Generation of CRISPR/Cas9-Based Mutants Supported by Fluorescent Seed Selection in Different Arabidopsis Accessions.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2484}, number = {}, pages = {161-182}, pmid = {35461452}, issn = {1940-6029}, mesh = {*Arabidopsis/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Plants, Genetically Modified/genetics ; RNA, Guide/genetics ; Seeds/genetics ; }, abstract = {Investigating the process of gamete formation in plants often requires the use of mutants of selected genes in various genetic backgrounds. For example, analysis of meiotic recombination based on sequencing or genotyping requires the generation of hybrids between two lines. Although T-DNA mutant collections of Arabidopsis thaliana are vast and easily accessible, they are largely confined to Col-0 background. This chapter describes how to efficiently generate knock-out mutants in different Arabidopsis accessions using CRISPR/Cas9 technology. The presented system is based on designing two single-guide RNAs (sgRNAs), which direct the Cas9 endonuclease to generate double-strand breaks at two sites, leading to genomic deletion in targeted gene. The presence of seed-expressed dsRed fluorescence cassette in the CRISPR construct facilitates preselection of genome-edited and transgene-free plants by monitoring the seed fluorescence under the epifluorescent microscope. The protocol provides the detailed information about all steps required to perform genome editing and to obtain loss-of-function mutants in different Arabidopsis accessions within merely two generations.}, }
@article {pmid35461116, year = {2022}, author = {Li, Y and Deng, F and Goldys, EM}, title = {A simple and versatile CRISPR/Cas12a-based immunosensing platform: Towards attomolar level sensitivity for small protein diagnostics.}, journal = {Talanta}, volume = {246}, number = {}, pages = {123469}, doi = {10.1016/j.talanta.2022.123469}, pmid = {35461116}, issn = {1873-3573}, abstract = {Recent advances in CRISPR/Cas biosensing have led to impressive performance in sensitivity, specificity, and speed for nucleic acid detection. However, the remarkable advantages (such as universality, ultralow, attomolar detection limits) of CRISPR/Cas biosensing systems are limited in testing non-nucleic acid targets. Herein, by synthesizing a functional hybrid conjugate of antibody and single strand DNA oligonucleotide, we had successfully demonstrated the capability to integrate CRISPR/Cas12a-based signal amplification into different types of immunoassay schemes without the need for any additional recognition molecule or molecular synthesis during the detection process, thus providing a simple but generally applicable approach to improve the conventional immunoassays with attomolar sensitivity for small protein detections, referred as the CRISPR-based Universal Immunoassay Signal Enhancer (CRUISE). CRUISE is capable of being integrated into various immunoassays either through the primary antibody or the secondary antibody, with sensitivity down to 1 fg mL-1 (∼50 aM) and 6 logs of linear range for detecting cytokines, such as IFN-γ and EGFR, under 3-4 h. It has a 103 times higher sensitivity compared to a commercial IFN-γ ELISA kit, but uses the same experimental scheme. The same 1 fg mL-1 sensitivity along with 6 logs of linear range was realized for IFN-γ detection in human plasma samples. We are expecting that our CRUISE provides an alternative but simple, user-friendly and effective strategy for those who rely on the use of immunoassays, while struggling with the limits of their sensitivity or detection ranges.}, }
@article {pmid35460444, year = {2022}, author = {Das, D and Singha, DL and Paswan, RR and Chowdhury, N and Sharma, M and Reddy, PS and Chikkaputtaiah, C}, title = {Recent advancements in CRISPR/Cas technology for accelerated crop improvement.}, journal = {Planta}, volume = {255}, number = {5}, pages = {109}, pmid = {35460444}, issn = {1432-2048}, support = {MLP-0007//Council of Scientific and Industrial Research, India/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; Genome, Plant/genetics ; *Plant Breeding/methods ; Plants, Genetically Modified/genetics ; Technology ; }, abstract = {MAIN CONCLUSION: Precise genome engineering approaches could be perceived as a second paradigm for targeted trait improvement in crop plants, with the potential to overcome the constraints imposed by conventional CRISPR/Cas technology. The likelihood of reduced agricultural production due to highly turbulent climatic conditions increases as the global population expands. The second paradigm of stress-resilient crops with enhanced tolerance and increased productivity against various stresses is paramount to support global production and consumption equilibrium. Although traditional breeding approaches have substantially increased crop production and yield, effective strategies are anticipated to restore crop productivity even further in meeting the world's increasing food demands. CRISPR/Cas, which originated in prokaryotes, has surfaced as a coveted genome editing tool in recent decades, reshaping plant molecular biology in unprecedented ways and paving the way for engineering stress-tolerant crops. CRISPR/Cas is distinguished by its efficiency, high target specificity, and modularity, enables precise genetic modification of crop plants, allowing for the creation of allelic variations in the germplasm and the development of novel and more productive agricultural practices. Additionally, a slew of advanced biotechnologies premised on the CRISPR/Cas methodologies have augmented fundamental research and plant synthetic biology toolkits. Here, we describe gene editing tools, including CRISPR/Cas and its imitative tools, such as base and prime editing, multiplex genome editing, chromosome engineering followed by their implications in crop genetic improvement. Further, we comprehensively discuss the latest developments of CRISPR/Cas technology including CRISPR-mediated gene drive, tissue-specific genome editing, dCas9 mediated epigenetic modification and programmed self-elimination of transgenes in plants. Finally, we highlight the applicability and scope of advanced CRISPR-based techniques in crop genetic improvement.}, }
@article {pmid35458632, year = {2022}, author = {Vojnits, K and Nakanishi, M and Porras, D and Kim, Y and Feng, Z and Golubeva, D and Bhatia, M}, title = {Developing CRISPR/Cas9-Mediated Fluorescent Reporter Human Pluripotent Stem-Cell Lines for High-Content Screening.}, journal = {Molecules (Basel, Switzerland)}, volume = {27}, number = {8}, pages = {}, pmid = {35458632}, issn = {1420-3049}, mesh = {*CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Cell Line ; Gene Editing/methods ; Genes, Reporter ; Green Fluorescent Proteins ; Humans ; *Pluripotent Stem Cells ; }, abstract = {Application of the CRISPR/Cas9 system to knock in fluorescent proteins to endogenous genes of interest in human pluripotent stem cells (hPSCs) has the potential to facilitate hPSC-based disease modeling, drug screening, and optimization of transplantation therapy. To evaluate the capability of fluorescent reporter hPSC lines for high-content screening approaches, we targeted EGFP to the endogenous OCT4 locus. Resulting hPSC-OCT4-EGFP lines generated expressed EGFP coincident with pluripotency markers and could be adapted to multi-well formats for high-content screening (HCS) campaigns. However, after long-term culture, hPSCs transiently lost their EGFP expression. Alternatively, through EGFP knock-in to the AAVS1 locus, we established a stable and consistent EGFP-expressing hPSC-AAVS1-EGFP line that maintained EGFP expression during in vitro hematopoietic and neural differentiation. Thus, hPSC-AAVS1-EGFP-derived sensory neurons could be adapted to a high-content screening platform that can be applied to high-throughput small-molecule screening and drug discovery campaigns. Our observations are consistent with recent findings indicating that high-frequency on-target complexities appear following CRISPR/Cas9 genome editing at the OCT4 locus. In contrast, we demonstrate that the AAVS1 locus is a safe genomic location in hPSCs with high gene expression that does not impact hPSC quality and differentiation. Our findings suggest that the CRISPR/Cas9-integrated AAVS1 system should be applied for generating stable reporter hPSC lines for long-term HCS approaches, and they underscore the importance of careful evaluation and selection of the applied reporter cell lines for HCS purposes.}, }
@article {pmid35458562, year = {2022}, author = {Liu, J and Tao, D and Chen, X and Shen, L and Zhu, L and Xu, B and Liu, H and Zhao, S and Li, X and Liu, X and Xie, S and Niu, L}, title = {Detection of Four Porcine Enteric Coronaviruses Using CRISPR-Cas12a Combined with Multiplex Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay.}, journal = {Viruses}, volume = {14}, number = {4}, pages = {}, pmid = {35458562}, issn = {1999-4915}, support = {No. 32072685//the Natural Science Foundation of China/ ; No. SCSZTD-2021-09//the Sichuan Science and Technology Support Program/ ; No. CARS-36-05B//the earmarked fund for China Agriculture Research System/ ; }, mesh = {Alphacoronavirus ; Animals ; CRISPR-Cas Systems ; *Coronavirus/genetics ; *Coronavirus Infections/diagnosis/genetics/veterinary ; Molecular Diagnostic Techniques ; Nucleic Acid Amplification Techniques ; *Porcine epidemic diarrhea virus/genetics ; RNA-Directed DNA Polymerase/genetics ; Sensitivity and Specificity ; Swine ; *Swine Diseases ; }, abstract = {Porcine enteric coronaviruses have caused immense economic losses to the global pig industry, and pose a potential risk for cross-species transmission. The clinical symptoms of the porcine enteric coronaviruses (CoVs) are similar, making it difficult to distinguish between the specific pathogens by symptoms alone. Here, a multiplex nucleic acid detection platform based on CRISPR/Cas12a and multiplex reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) was developed for the detection of four diarrhea CoVs: porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV). With this strategy, we realized a visual colorimetric readout visible to the naked eye without specialized instrumentation by using a ROX-labeled single-stranded DNA-fluorescence-quenched (ssDNA-FQ) reporter. Our method achieved single-copy sensitivity with no cross-reactivity in the identification and detection of the target viruses. In addition, we successfully detected these four enteric CoVs from RNA of clinical samples. Thus, we established a rapid, sensitive, and on-site multiplex molecular differential diagnosis technology for porcine enteric CoVs.}, }
@article {pmid35458523, year = {2022}, author = {Calderón, K and Rojas-Neyra, A and Carbajal-Lévano, B and Luján-Valenzuela, L and Ticona, J and Isasi-Rivas, G and Montalvan, A and Criollo-Orozco, M and Huaccachi-Gonzáles, E and Tataje-Lavanda, L and Alvarez, KLF and Fernández-Sánchez, M and Fernández-Díaz, M and Tang, N and Yao, Y and Nair, V}, title = {A Recombinant Turkey Herpesvirus Expressing the F Protein of Newcastle Disease Virus Genotype XII Generated by NHEJ-CRISPR/Cas9 and Cre-LoxP Systems Confers Protection against Genotype XII Challenge in Chickens.}, journal = {Viruses}, volume = {14}, number = {4}, pages = {}, pmid = {35458523}, issn = {1999-4915}, support = {BBS/E/I/00007039/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; Contract N° 038-INNOVATEPERU-PIEC1-2020//Innovate Peru Program of the Ministry of Production/ ; BBS/E/I/00007039//Biotechnology and Biological Sciences Research Council (BBSRC)/ ; }, mesh = {Animals ; Antibodies, Viral ; CRISPR-Cas Systems ; Chickens ; Genotype ; Herpesvirus 1, Meleagrid/genetics ; *Herpesvirus 2, Gallid ; Integrases ; *Newcastle Disease/prevention &amp; control ; Newcastle disease virus/genetics ; *Poultry Diseases ; Vaccines, Synthetic/genetics ; *Viral Vaccines/genetics ; }, abstract = {In this study, we developed a new recombinant virus rHVT-F using a Turkey herpesvirus (HVT) vector, expressing the fusion (F) protein of the genotype XII Newcastle disease virus (NDV) circulating in Peru. We evaluated the viral shedding and efficacy against the NDV genotype XII challenge in specific pathogen-free (SPF) chickens. The F protein expression cassette was inserted in the unique long (UL) UL45-UL46 intergenic locus of the HVT genome by utilizing a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 gene-editing technology via a non-homologous end joining (NHEJ) repair pathway. The rHVT-F virus, which expressed the F protein stably in vitro and in vivo, showed similar growth kinetics to the wild-type HVT (wtHVT) virus. The F protein expression of the rHVT-F virus was detected by an indirect immunofluorescence assay (IFA), Western blotting, and a flow cytometry assay. The presence of an NDV-specific IgY antibody was detected in serum samples by an enzyme-linked immunosorbent assay (ELISA) in SPF chickens vaccinated with the rHVT-F virus. In the challenge experiment, the rHVT-F vaccine fully protects a high, and significantly reduced, virus shedding in oral at 5 days post-challenge (dpc). In conclusion, this new rHVT-F vaccine candidate is capable of fully protecting SPF chickens against the genotype XII challenge.}, }
@article {pmid35457940, year = {2022}, author = {Song, W and Zhang, T and Lin, H and Yang, Y and Zhao, G and Huang, X}, title = {Conventional and Microfluidic Methods for the Detection of Nucleic Acid of SARS-CoV-2.}, journal = {Micromachines}, volume = {13}, number = {4}, pages = {}, pmid = {35457940}, issn = {2072-666X}, abstract = {Nucleic acid testing (NAT) played a crucial role in containing the spread of SARS-CoV-2 during the epidemic. The gold standard technique, the quantitative real-time polymerase chain reaction (qRT-PCR) technique, is currently used by the government and medical boards to detect SARS-CoV-2. Due to the limitations of this technology, it is not capable of meeting the needs of large-scale rapid detection. To solve this problem, many new techniques for detecting nucleic acids of SARS-CoV-2 have been reported. Therefore, a review that systematically and comprehensively introduces and compares various detection technologies is needed. In this paper, we not only review the traditional NAT but also provide an overview of microfluidic-based NAT technologies and summarize and discuss the characteristics and development prospects of these techniques.}, }
@article {pmid35457271, year = {2022}, author = {Zegeye, WA and Tsegaw, M and Zhang, Y and Cao, L}, title = {CRISPR-Based Genome Editing: Advancements and Opportunities for Rice Improvement.}, journal = {International journal of molecular sciences}, volume = {23}, number = {8}, pages = {}, pmid = {35457271}, issn = {1422-0067}, mesh = {CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; *Gene Editing ; Genome, Plant ; *Oryza/genetics ; Plant Breeding ; Plants, Genetically Modified/genetics ; }, abstract = {To increase the potentiality of crop production for future food security, new technologies for plant breeding are required, including genome editing technology-being one of the most promising. Genome editing with the CRISPR/Cas system has attracted researchers in the last decade as a safer and easier tool for genome editing in a variety of living organisms including rice. Genome editing has transformed agriculture by reducing biotic and abiotic stresses and increasing yield. Recently, genome editing technologies have been developed quickly in order to avoid the challenges that genetically modified crops face. Developing transgenic-free edited plants without introducing foreign DNA has received regulatory approval in a number of countries. Several ongoing efforts from various countries are rapidly expanding to adopt the innovations. This review covers the mechanisms of CRISPR/Cas9, comparisons of CRISPR/Cas9 with other gene-editing technologies-including newly emerged Cas variants-and focuses on CRISPR/Cas9-targeted genes for rice crop improvement. We have further highlighted CRISPR/Cas9 vector construction model design and different bioinformatics tools for target site selection.}, }
@article {pmid35457234, year = {2022}, author = {Rozov, SM and Permyakova, NV and Sidorchuk, YV and Deineko, EV}, title = {Optimization of Genome Knock-In Method: Search for the Most Efficient Genome Regions for Transgene Expression in Plants.}, journal = {International journal of molecular sciences}, volume = {23}, number = {8}, pages = {}, pmid = {35457234}, issn = {1422-0067}, support = {21-14-00091//Russian Science Foundation/ ; }, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; Genome, Plant ; Mammals/genetics ; Plants, Genetically Modified/genetics ; Recombinant Proteins/genetics ; Transgenes ; }, abstract = {Plant expression systems are currently regarded as promising alternative platforms for the production of recombinant proteins, including the proteins for biopharmaceutical purposes. However, the accumulation level of a target protein in plant expression systems is still rather low compared with the other existing systems, namely, mammalian, yeast, and E. coli cells. To solve this problem, numerous methods and approaches have been designed and developed. At the same time, the random nature of the distribution of transgenes over the genome can lead to gene silencing, variability in the accumulation of recombinant protein, and also to various insertional mutations. The current research study considered inserting target genes into pre-selected regions of the plant genome (genomic "safe harbors") using the CRISPR/Cas system. Regions of genes expressed constitutively and at a high transcriptional level in plant cells (housekeeping genes) that are of interest as attractive targets for the delivery of target genes were characterized. The results of the first attempts to deliver target genes to the regions of housekeeping genes are discussed. The approach of "euchromatization" of the transgene integration region using the modified dCas9 associated with transcription factors is considered. A number of the specific features in the spatial chromatin organization allowing individual genes to efficiently transcribe are discussed.}, }
@article {pmid35457228, year = {2022}, author = {López-Márquez, A and Morín, M and Fernández-Peñalver, S and Badosa, C and Hernández-Delgado, A and Natera-de Benito, D and Ortez, C and Nascimento, A and Grinberg, D and Balcells, S and Roldán, M and Moreno-Pelayo, MÁ and Jiménez-Mallebrera, C}, title = {CRISPR/Cas9-Mediated Allele-Specific Disruption of a Dominant COL6A1 Pathogenic Variant Improves Collagen VI Network in Patient Fibroblasts.}, journal = {International journal of molecular sciences}, volume = {23}, number = {8}, pages = {}, pmid = {35457228}, issn = {1422-0067}, support = {PI19/0122//Instituto de Salud Carlos III/ ; IMP/00009//Instituto de Salud Carlos III/ ; ER19P5AC728/2021//Centre for Biomedical Network Research on Rare Diseases/ ; ACCI2018-02//Centre for Biomedical Network Research on Rare Diseases/ ; CAM, B2017/ BMD3721//Comunidad de Madrid/ ; }, mesh = {Alleles ; *CRISPR-Cas Systems/genetics ; *Collagen Type VI/genetics/metabolism ; Extracellular Matrix/metabolism ; Fibroblasts/metabolism ; Humans ; Mutation ; }, abstract = {Collagen VI-related disorders are the second most common congenital muscular dystrophies for which no treatments are presently available. They are mostly caused by dominant-negative pathogenic variants in the genes encoding α chains of collagen VI, a heteromeric network forming collagen; for example, the c.877G>A; p.Gly293Arg COL6A1 variant, which alters the proper association of the tetramers to form microfibrils. We tested the potential of CRISPR/Cas9-based genome editing to silence or correct (using a donor template) a mutant allele in the dermal fibroblasts of four individuals bearing the c.877G>A pathogenic variant. Evaluation of gene-edited cells by next-generation sequencing revealed that correction of the mutant allele by homologous-directed repair occurred at a frequency lower than 1%. However, the presence of frameshift variants and others that provoked the silencing of the mutant allele were found in >40% of reads, with no effects on the wild-type allele. This was confirmed by droplet digital PCR with allele-specific probes, which revealed a reduction in the expression of the mutant allele. Finally, immunofluorescence analyses revealed a recovery in the collagen VI extracellular matrix. In summary, we demonstrate that CRISPR/Cas9 gene-edition can specifically reverse the pathogenic effects of a dominant negative variant in COL6A1.}, }
@article {pmid35457099, year = {2022}, author = {Vanhoye, X and Janin, A and Caillaud, A and Rimbert, A and Venet, F and Gossez, M and Dijk, W and Marmontel, O and Nony, S and Chatelain, C and Durand, C and Lindenbaum, P and Rieusset, J and Cariou, B and Moulin, P and Di Filippo, M}, title = {APOB CRISPR-Cas9 Engineering in Hypobetalipoproteinemia: A Promising Tool for Functional Studies of Novel Variants.}, journal = {International journal of molecular sciences}, volume = {23}, number = {8}, pages = {}, pmid = {35457099}, issn = {1422-0067}, support = {Young Researcher Project//Hospices Civils de Lyon/ ; ANR-16-RHUS-0007//Agence Nationale de la Recherche/ ; Fondation Recherche Médicale//Fondation Recherche Médicale/ ; }, mesh = {Apolipoproteins B/metabolism ; CRISPR-Cas Systems ; *Fatty Liver/genetics ; Humans ; *Hypobetalipoproteinemia, Familial, Apolipoprotein B/genetics ; *Hypobetalipoproteinemias/diagnosis/genetics/metabolism ; }, abstract = {Hypobetalipoproteinemia is characterized by LDL-cholesterol and apolipoprotein B (apoB) plasma levels below the fifth percentile for age and sex. Familial hypobetalipoproteinemia (FHBL) is mostly caused by premature termination codons in the APOB gene, a condition associated with fatty liver and steatohepatitis. Nevertheless, many families with a FHBL phenotype carry APOB missense variants of uncertain significance (VUS). We here aimed to develop a proof-of-principle experiment to assess the pathogenicity of VUS using the genome editing of human liver cells. We identified a novel heterozygous APOB-VUS (p.Leu351Arg), in a FHBL family. We generated APOB knock-out (KO) and APOB-p.Leu351Arg knock-in Huh7 cells using CRISPR-Cas9 technology and studied the APOB expression, synthesis and secretion by digital droplet PCR and ELISA quantification. The APOB expression was decreased by 70% in the heterozygous APOB-KO cells and almost abolished in the homozygous-KO cells, with a consistent decrease in apoB production and secretion. The APOB-p.Leu351Arg homozygous cells presented with a 40% decreased APOB expression and undetectable apoB levels in cellular extracts and supernatant. Thus, the p.Leu351Arg affected the apoB secretion, which led us to classify this new variant as likely pathogenic and to set up a hepatic follow-up in this family. Therefore, the functional assessment of APOB-missense variants, using gene-editing technologies, will lead to improvements in the molecular diagnosis of FHBL and the personalized follow-up of these patients.}, }
@article {pmid35456898, year = {2022}, author = {Dunbar, T and Tsakirpaloglou, N and Septiningsih, EM and Thomson, MJ}, title = {Carbon Nanotube-Mediated Plasmid DNA Delivery in Rice Leaves and Seeds.}, journal = {International journal of molecular sciences}, volume = {23}, number = {8}, pages = {}, pmid = {35456898}, issn = {1422-0067}, support = {2020-67013-31811//United States Department of Agriculture/ ; }, mesh = {CRISPR-Cas Systems/genetics ; DNA ; Gene Editing/methods ; Genome, Plant ; *Nanotubes, Carbon ; *Oryza/genetics ; Plant Leaves/genetics ; Plants/genetics ; Plants, Genetically Modified/genetics ; Plasmids/genetics ; Seeds/genetics ; }, abstract = {CRISPR-Cas gene editing technologies offer the potential to modify crops precisely; however, in vitro plant transformation and regeneration techniques present a bottleneck due to the lengthy and genotype-specific tissue culture process. Ideally, in planta transformation can bypass tissue culture and directly lead to transformed plants, but efficient in planta delivery and transformation remains a challenge. This study investigates transformation methods that have the potential to directly alter germline cells, eliminating the challenge of in vitro plant regeneration. Recent studies have demonstrated that carbon nanotubes (CNTs) loaded with plasmid DNA can diffuse through plant cell walls, facilitating transient expression of foreign genetic elements in plant tissues. To test if this approach is a viable technique for in planta transformation, CNT-mediated plasmid DNA delivery into rice tissues was performed using leaf and excised-embryo infiltration with reporter genes. Quantitative and qualitative data indicate that CNTs facilitate plasmid DNA delivery in rice leaf and embryo tissues, resulting in transient GFP, YFP, and GUS expression. Experiments were also initiated with CRISPR-Cas vectors targeting the phytoene desaturase (PDS) gene for CNT delivery into mature embryos to create heritable genetic edits. Overall, the results suggest that CNT-based delivery of plasmid DNA appears promising for in planta transformation, and further optimization can enable high-throughput gene editing to accelerate functional genomics and crop improvement activities.}, }
@article {pmid35453874, year = {2022}, author = {Li, Y and Shi, Z and Hu, A and Cui, J and Yang, K and Liu, Y and Deng, G and Zhu, C and Zhu, L}, title = {Rapid One-Tube RPA-CRISPR/Cas12 Detection Platform for Methicillin-Resistant Staphylococcus aureus.}, journal = {Diagnostics (Basel, Switzerland)}, volume = {12}, number = {4}, pages = {}, pmid = {35453874}, issn = {2075-4418}, support = {82002189//National Natural Science Foundation of China/ ; 2008085QF311//Natural Science Foundation of Anhui Province of China/ ; 202004d07020014//The Key Research and Development Program of Anhui Province of China/ ; 2018489, 2021442//Youth Innovation Promotion Association of the Chinese Academy of Sciences/ ; YZJJ2020QN35, YZJJ2021QN03//Dean's Fund of Hefei Institute of Physical Science, China/ ; }, abstract = {Methicillin-resistant Staphylococcus aureus (MRSA) is a severe health threat causing high-level morbidity and mortality in health care environments and in community settings. Though existing diagnostic methods, including PCR and culture-based methods, are routinely used in clinical practice, they are not appropriate for rapid point-of-care testing (POCT). Recently, since the development of the CRISPR/Cas technology, new possibilities for rapid point-of-care detection have emerged. In this study, we developed a rapid, accurate, and contamination-free platform for MRSA detection by integrating recombinase polymerase amplification (RPA) with the Cas12 system into one tube. Using this approach, visual MRSA detection could be achieved in 20 min. Based on the one-tube RPA-CRISPR/Cas12a platform, the assay results are visualized by lateral flow test strips (LFS) and fluorescent-based methods, including real-time and end-point fluorescence. This platform allows specific MRSA detection with a sensitivity of 10 copies for the fluorescence method and a range of 10-100 copies for the LFS. The results of 23 samples from clinical MRSA isolates showed that the coincidence rate was 100% and 95.7% of the fluorescence method and LFS, respectively, compared to qPCR. In conclusion, the one-tube RPA-CRISPR/Cas12a platform is an effective method for MRSA detection with significant potential in future practical POCT applications.}, }
@article {pmid35453665, year = {2022}, author = {Azlan, A and Rajasegaran, Y and Kang Zi, K and Rosli, AA and Yik, MY and Yusoff, NM and Heidenreich, O and Moses, EJ}, title = {Elucidating miRNA Function in Cancer Biology via the Molecular Genetics' Toolbox.}, journal = {Biomedicines}, volume = {10}, number = {4}, pages = {}, pmid = {35453665}, issn = {2227-9059}, support = {FRGS/1/2018/SKK08/USM/02/8 (203/CIPPT/6711672)//Ministry of Higher Education/ ; USM-RUI: 1001/CIPPT/8012265//Universiti Sains Malaysia/ ; }, abstract = {Micro-RNA (miRNAs) are short non-coding RNAs of about 18-20 nucleotides in length and are implicated in many cellular processes including proliferation, development, differentiation, apoptosis and cell signaling. Furthermore, it is well known that miRNA expression is frequently dysregulated in many cancers. Therefore, this review will highlight the various mechanisms by which microRNAs are dysregulated in cancer. Further highlights include the abundance of molecular genetics tools that are currently available to study miRNA function as well as their advantages and disadvantages with a special focus on various CRISPR/Cas systems This review provides general workflows and some practical considerations when studying miRNA function thus enabling researchers to make informed decisions in regards to the appropriate molecular genetics tool to be utilized for their experiments.}, }
@article {pmid35452778, year = {2022}, author = {Ceasar, SA and Maharajan, T and Hillary, VE and Ajeesh Krishna, TP}, title = {Insights to improve the plant nutrient transport by CRISPR/Cas system.}, journal = {Biotechnology advances}, volume = {59}, number = {}, pages = {107963}, doi = {10.1016/j.biotechadv.2022.107963}, pmid = {35452778}, issn = {1873-1899}, abstract = {We need to improve food production to feed the ever growing world population especially in a changing climate. Nutrient deficiency in soils is one of the primary bottlenecks affecting the crop production both in developed and developing countries. Farmers are forced to apply synthetic fertilizers to improve the crop production to meet the demand. Understanding the mechanism of nutrient transport is helpful to improve the nutrient-use efficiency of crops and promote the sustainable agriculture. Many transporters involved in the acquisition, export and redistribution of nutrients in plants are characterized. In these studies, heterologous systems like yeast and Xenopus were most frequently used to study the transport function of plant nutrient transporters. CRIPSR/Cas system introduced recently has taken central stage for efficient genome editing in diverse organisms including plants. In this review, we discuss the key nutrient transporters involved in the acquisition and redistribution of nutrients from soil. We draw insights on the possible application CRISPR/Cas system for improving the nutrient transport in plants by engineering key residues of nutrient transporters, transcriptional regulation of nutrient transport signals, engineering motifs in promoters and transcription factors. CRISPR-based engineering of plant nutrient transport not only helps to study the process in native plants with conserved regulatory system but also aid to develop non-transgenic crops with better nutrient use-efficiency. This will reduce the application of synthetic fertilizers and promote the sustainable agriculture strengthening the food and nutrient security.}, }
@article {pmid35452274, year = {2022}, author = {Anliker, B and Childs, L and Rau, J and Renner, M and Schüle, S and Schuessler-Lenz, M and Sebe, A}, title = {Regulatory Considerations for Clinical Trial Applications with CRISPR-Based Medicinal Products.}, journal = {The CRISPR journal}, volume = {}, number = {}, pages = {}, doi = {10.1089/crispr.2021.0148}, pmid = {35452274}, issn = {2573-1602}, abstract = {Since first proposed as a new tool for gene targeting and genome editing, CRISPR technology has quickly advanced into the clinical stage. Initial studies highlight the potential for CRISPR-Cas9-mediated therapeutic approaches in human medicine to correct incurable genetic diseases and enhance cell-based therapeutic approaches. While acknowledging the opportunities this technology brings for the treatment of patients with severe diseases, timely development of these innovative medicinal products requires regulatory oversight and adaptation of regulatory requirements to ensure the safety and efficacy of medicinal products based on CRISPR technology. We briefly present the current regulatory framework applicable for CRISPR-Cas-based developments as advanced therapy medicinal products. Moreover, scientific- and regulatory-driven considerations relevant for advancing product development toward clinical trial applications in Germany are highlighted by discussing the key aspects of quality and nonclinical and clinical development requirements.}, }
@article {pmid35452075, year = {2022}, author = {Oh, SA and Senger, K and Madireddi, S and Akhmetzyanova, I and Ishizuka, IE and Tarighat, S and Lo, JH and Shaw, D and Haley, B and Rutz, S}, title = {High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA.}, journal = {The Journal of experimental medicine}, volume = {219}, number = {5}, pages = {}, pmid = {35452075}, issn = {1540-9538}, mesh = {*CRISPR-Cas Systems/genetics ; DNA/genetics ; *Gene Editing/methods ; Humans ; Plasmids/genetics ; T-Lymphocytes ; }, abstract = {Genome engineering of T lymphocytes, the main effectors of antitumor adaptive immune responses, has the potential to uncover unique insights into their functions and enable the development of next-generation adoptive T cell therapies. Viral gene delivery into T cells, which is currently used to generate CAR T cells, has limitations in regard to targeting precision, cargo flexibility, and reagent production. Nonviral methods for effective CRISPR/Cas9-mediated gene knock-out in primary human T cells have been developed, but complementary techniques for nonviral gene knock-in can be cumbersome and inefficient. Here, we report a convenient and scalable nonviral method that allows precise gene edits and transgene integration in primary human T cells, using plasmid donor DNA template and Cas9-RNP. This method is highly efficient for single and multiplex gene manipulation, without compromising T cell function, and is thus valuable for use in basic and translational research.}, }
@article {pmid35451949, year = {2022}, author = {Han, X and Zhou, X and Pei, Z and Stanton, C and Ross, RP and Zhao, J and Zhang, H and Yang, B and Chen, W}, title = {Characterization of CRISPR-Cas systems in Bifidobacterium breve.}, journal = {Microbial genomics}, volume = {8}, number = {4}, pages = {}, doi = {10.1099/mgen.0.000812}, pmid = {35451949}, issn = {2057-5858}, mesh = {*Bifidobacterium breve/genetics ; *CRISPR-Cas Systems/genetics ; DNA ; Sequence Analysis, DNA ; }, abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) system is an important adaptive immune system for bacteria to resist foreign DNA infection, which has been widely used in genotyping and gene editing. To provide a theoretical basis for the application of the CRISPR-Cas system in Bifidobacterium breve, the occurrence and diversity of CRISPR-Cas systems were analysed in 150 B. breve strains. Specifically, 47 % (71/150) of B. breve genomes possessed the CRISPR-Cas system, and type I-C CRISPR-Cas system was the most widely distributed among those strains. The spacer sequences present in B. breve can be used as a genotyping marker. Additionally, the phage assembly-related proteins were important targets of the type I-C CRISPR-Cas system in B. breve, and the protospacer adjacent motif sequences were further characterized in B. breve type I-C system as 5'-TTC-3'. All these results might provide a molecular basis for the development of endogenous genome editing tools in B. breve.}, }
@article {pmid35451838, year = {2022}, author = {Luo, T and Li, J and He, Y and Liu, H and Deng, Z and Long, X and Wan, Q and Ding, J and Gong, Z and Yang, Y and Zhong, S}, title = {Designing a CRISPR/Cas12a- and Au-Nanobeacon-Based Diagnostic Biosensor Enabling Direct, Rapid, and Sensitive miRNA Detection.}, journal = {Analytical chemistry}, volume = {94}, number = {17}, pages = {6566-6573}, doi = {10.1021/acs.analchem.2c00401}, pmid = {35451838}, issn = {1520-6882}, mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; DNA/genetics ; DNA, Single-Stranded/genetics ; *MicroRNAs/genetics ; }, abstract = {Direct, rapid, sensitive, and selective detection of nucleic acids in complex biological fluids is crucial for medical early diagnosis. We herein combine the trans-cleavage ability of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a with Au-nanobeacon to establish a CRISPR-based biosensor, providing rapid miRNA detection with high speed and attomolar sensitivity. In this strategy, we first report that the trans-cleavage activity of CRISPR/cas12a, which was previously reported to be triggered only by target ssDNA or dsDNA, can be activated by the target miRNA directly. Therefore, this method is direct, i.e., does not need the conversion of miRNA into its complementary DNA (cDNA). Meanwhile, as compared to the traditional ssDNA reporters and molecular beacon (MB) reporters, the Au-nanobeacon reporters exhibit improved reaction kinetics and sensitivity. In this assay, the miRNA-21 could be detected with very high sensitivity in only 5 min. Finally, the proposed strategy enables rapid, sensitive, and selective miRNA determination in complex biological samples, providing a potential tool for medical early diagnosis.}, }
@article {pmid35451132, year = {2022}, author = {Kershanskaya, OI and Yessenbaeva, GL and Nelidova, DS and Karabekova, AN and Sadullaeva, ZN}, title = {CRISPR/Cas genome editing perspectives for barley breeding.}, journal = {Physiologia plantarum}, volume = {}, number = {}, pages = {e13686}, doi = {10.1111/ppl.13686}, pmid = {35451132}, issn = {1399-3054}, abstract = {The CRISPR/Cas9 technology shows potential to improve crop breeding efficiency and antiviral defense. The interest in DNA editing in crops has grown due to the possibility of increasing the resistance of different plants to many viruses. Our aim was to create an elite disease-resistant local barley cultivar using CRISPR/Cas9 biotechnology. For this purpose, we used CRISPR/Cas 9-eIF4E with the eukaryotic translation initiation factor 4E (eIF4E) barley gene to edit the genomes of five local Kazakhstan barley cultivars. After identifying the single guide RNA (sgRNA) target sequences, they were synthesized and cloned into the CRISPR-plant vector before being introduced into barley cells via our own patented Agrobacterium germ-line transformation technique. Barley plants eIF4E-modified were successfully obtained and were resistant to virus infection. Based on our research, the CRISPR/Cas9 system for plant genome editing could be a prospect for applying this breakthrough biotechnology in barley breeding. This article is protected by copyright. All rights reserved.}, }
@article {pmid35448780, year = {2022}, author = {Haroon, M and Wang, X and Afzal, R and Zafar, MM and Idrees, F and Batool, M and Khan, AS and Imran, M}, title = {Novel Plant Breeding Techniques Shake Hands with Cereals to Increase Production.}, journal = {Plants (Basel, Switzerland)}, volume = {11}, number = {8}, pages = {}, pmid = {35448780}, issn = {2223-7747}, support = {2018JQ5218//National Natural Science Foundation of China/ ; }, abstract = {Cereals are the main source of human food on our planet. The ever-increasing food demand, continuously changing environment, and diseases of cereal crops have made adequate production a challenging task for feeding the ever-increasing population. Plant breeders are striving their hardest to increase production by manipulating conventional breeding methods based on the biology of plants, either self-pollinating or cross-pollinating. However, traditional approaches take a decade, space, and inputs in order to make crosses and release improved varieties. Recent advancements in genome editing tools (GETs) have increased the possibility of precise and rapid genome editing. New GETs such as CRISPR/Cas9, CRISPR/Cpf1, prime editing, base editing, dCas9 epigenetic modification, and several other transgene-free genome editing approaches are available to fill the lacuna of selection cycles and limited genetic diversity. Over the last few years, these technologies have led to revolutionary developments and researchers have quickly attained remarkable achievements. However, GETs are associated with various bottlenecks that prevent the scaling development of new varieties that can be dealt with by integrating the GETs with the improved conventional breeding methods such as speed breeding, which would take plant breeding to the next level. In this review, we have summarized all these traditional, molecular, and integrated approaches to speed up the breeding procedure of cereals.}, }
@article {pmid35448772, year = {2022}, author = {Niazian, M and Belzile, F and Torkamaneh, D}, title = {CRISPR/Cas9 in Planta Hairy Root Transformation: A Powerful Platform for Functional Analysis of Root Traits in Soybean.}, journal = {Plants (Basel, Switzerland)}, volume = {11}, number = {8}, pages = {}, pmid = {35448772}, issn = {2223-7747}, support = {6548//Genome Canada/ ; }, abstract = {Sequence and expression data obtained by next-generation sequencing (NGS)-based forward genetics methods often allow the identification of candidate causal genes. To provide true experimental evidence of a gene's function, reverse genetics techniques are highly valuable. Site-directed mutagenesis through transfer DNA (T-DNA) delivery is an efficient reverse screen method in plant functional analysis. Precise modification of targeted crop genome sequences is possible through the stable and/or transient delivery of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (CRISPR/Cas) reagents. Currently, CRISPR/Cas9 is the most powerful reverse genetics approach for fast and precise functional analysis of candidate genes/mutations of interest. Rapid and large-scale analyses of CRISPR/Cas-induced mutagenesis is achievable through Agrobacterium rhizogenes-mediated hairy root transformation. The combination of A. rhizogenes hairy root-CRISPR/Cas provides an extraordinary platform for rapid, precise, easy, and cost-effective "in root" functional analysis of genes of interest in legume plants, including soybean. Both hairy root transformation and CRISPR/Cas9 techniques have their own complexities and considerations. Here, we discuss recent advancements in soybean hairy root transformation and CRISPR/Cas9 techniques. We highlight the critical factors required to enhance mutation induction and hairy root transformation, including the new generation of reporter genes, methods of Agrobacterium infection, accurate gRNA design strategies, Cas9 variants, gene regulatory elements of gRNAs and Cas9 nuclease cassettes and their configuration in the final binary vector to study genes involved in root-related traits in soybean.}, }
@article {pmid35447246, year = {2022}, author = {Kanafi, MM and Tavallaei, M}, title = {Overview of advances in CRISPR/deadCas9 technology and its applications in human diseases.}, journal = {Gene}, volume = {830}, number = {}, pages = {146518}, doi = {10.1016/j.gene.2022.146518}, pmid = {35447246}, issn = {1879-0038}, abstract = {Prokaryotes possess an adaptive immune system using various CRISPR associated (Cas) genes to make an archive of records from invading phages and eliminate them upon re-exposure when specialized Cas proteins cut foreign DNA into small pieces. On the basis of the different types of Cas proteins, CRISPR systems seen in some prokaryotic genomes, are different to each other. It has been proved that CRISPR has a great potential for genome engineering. Studies have also demonstrated that in comparison to the preceding genome engineering tools CRISPR/Cas systems can be harnessed as a flexible tool with easy multiplexing and scaling ability. Recent studies suggest that CRISPR/Cas systems can also be used for non-genome engineering roles. Isolation and identification of new Cas proteins or modification of existing ones are effectively increasing the number of CRISPR applications and helps its development. D10A and H840A mutations at RuvC and HNH endonuclease domains of wild type Streptococcus pyogenes Cas9 (SpCas9) respectively creates a nuclease, dead Cas9 (dCas9) molecule, that does not cut target DNA but still retains its capability for binding to target DNA based on the gRNA targeting sequence. In this article we review the potentials of this enzyme, dCas9, toward development of the applications of CRISPR/dCas9 technology in fields such as; visualization of genomic loci, disease diagnosis and transcriptional repression and activation.}, }
@article {pmid35446558, year = {2022}, author = {Chen, Z and Devi, G and Arif, A and Zamore, PD and Sontheimer, EJ and Watts, JK}, title = {Tetrazine-Ligated CRISPR sgRNAs for Efficient Genome Editing.}, journal = {ACS chemical biology}, volume = {}, number = {}, pages = {}, doi = {10.1021/acschembio.2c00116}, pmid = {35446558}, issn = {1554-8937}, abstract = {CRISPR-Cas technology has revolutionized genome editing. Its broad and fast-growing application in biomedical research and therapeutics has led to increased demand for guide RNAs. The synthesis of chemically modified single-guide RNAs (sgRNAs) containing >100 nucleotides remains a bottleneck. Here we report the development of a tetrazine ligation method for the preparation of sgRNAs. A tetrazine moiety on the 3'-end of the crRNA and a norbornene moiety on the 5'-end of the tracrRNA enable successful ligation between crRNA and tracrRNA to form sgRNA under mild conditions. Tetrazine-ligated sgRNAs allow efficient genome editing of reporter and endogenous loci in human cells. High-efficiency editing requires structural optimization of the linker.}, }
@article {pmid35446391, year = {2022}, author = {Lind Gleerup, J and Mogensen, TH}, title = {CRISPR-Cas in diagnostics and therapy of infectious diseases.}, journal = {The Journal of infectious diseases}, volume = {}, number = {}, pages = {}, doi = {10.1093/infdis/jiac145}, pmid = {35446391}, issn = {1537-6613}, abstract = {Infectious diseases are a major threat to the global health. The rise in antimicrobial resistant organisms, incurable chronic infections and an increasing demand for rapid accurate diagnostics have prompted researchers to experiment with new approaches. Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR associated protein is a naturally occurring adaptive immune system in bacteria, that has been developed as a tool for performing genomic alterations in any genome of interest, including humans and microbes. Accordingly, several studies have been conducted to investigate how the technology can be utilized in infectious diseases to improve diagnostics, disrupt antimicrobial resistance, and cure chronic infections. This review will provide an overview of the CRISPR-Cas system, and how it has been applied in studies on infectious diseases. The review will also investigate the current challenges of the technology and the improvements that are needed for the platform to be adopted for clinical use in patients.}, }
@article {pmid35445018, year = {2022}, author = {Hu, Y and Li, W}, title = {Development and Application of CRISPR-Cas Based Tools.}, journal = {Frontiers in cell and developmental biology}, volume = {10}, number = {}, pages = {834646}, pmid = {35445018}, issn = {2296-634X}, abstract = {Abundant CRISPR-Cas systems in nature provide us with unlimited valuable resources to develop a variety of versatile tools, which are powerful weapons in biological discovery and disease treatment. Here, we systematically review the development of CRISPR-Cas based tools from DNA nuclease to RNA nuclease, from nuclease dependent-tools to nucleic acid recognition dependent-tools. Also, considering the limitations and challenges of current CRISPR-Cas based tools, we discuss the potential directions for development of novel CRISPR toolkits in the future.}, }
@article {pmid35442747, year = {2022}, author = {Zou, Y and Sun, X and Yang, Q and Zheng, M and Shimoni, O and Ruan, W and Wang, Y and Zhang, D and Yin, J and Huang, X and Tao, W and Park, JB and Liang, XJ and Leong, KW and Shi, B}, title = {Blood-brain barrier-penetrating single CRISPR-Cas9 nanocapsules for effective and safe glioblastoma gene therapy.}, journal = {Science advances}, volume = {8}, number = {16}, pages = {eabm8011}, pmid = {35442747}, issn = {2375-2548}, mesh = {Animals ; Blood-Brain Barrier ; CRISPR-Cas Systems ; Gene Editing ; Genetic Therapy ; *Glioblastoma/genetics/therapy ; Mice ; *Nanocapsules ; RNA, Guide/genetics ; }, abstract = {We designed a unique nanocapsule for efficient single CRISPR-Cas9 capsuling, noninvasive brain delivery and tumor cell targeting, demonstrating an effective and safe strategy for glioblastoma gene therapy. Our CRISPR-Cas9 nanocapsules can be simply fabricated by encapsulating the single Cas9/sgRNA complex within a glutathione-sensitive polymer shell incorporating a dual-action ligand that facilitates BBB penetration, tumor cell targeting, and Cas9/sgRNA selective release. Our encapsulating nanocapsules evidenced promising glioblastoma tissue targeting that led to high PLK1 gene editing efficiency in a brain tumor (up to 38.1%) with negligible (less than 0.5%) off-target gene editing in high-risk tissues. Treatment with nanocapsules extended median survival time (68 days versus 24 days in nonfunctional sgRNA-treated mice). Our new CRISPR-Cas9 delivery system thus addresses various delivery challenges to demonstrate safe and tumor-specific delivery of gene editing Cas9 ribonucleoprotein for improved glioblastoma treatment that may potentially be therapeutically useful in other brain diseases.}, }
@article {pmid35440677, year = {2022}, author = {de Souza Pacheco, I and Doss, AA and Vindiola, BG and Brown, DJ and Ettinger, CL and Stajich, JE and Redak, RA and Walling, LL and Atkinson, PW}, title = {Efficient CRISPR/Cas9-mediated genome modification of the glassy-winged sharpshooter Homalodisca vitripennis (Germar).}, journal = {Scientific reports}, volume = {12}, number = {1}, pages = {6428}, pmid = {35440677}, issn = {2045-2322}, support = {01170-002//California Department of Food and Agriculture/ ; 012604-002//Animal and Plant Health Inspection Service/ ; }, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Hemiptera/genetics ; Pigmentation/genetics ; }, abstract = {CRISPR/Cas9 technology enables the extension of genetic techniques into insect pests previously refractory to genetic analysis. We report the establishment of genetic analysis in the glassy-winged sharpshooter (GWSS), Homalodisca vitripennis, which is a significant leafhopper pest of agriculture in California. We use a novel and simple approach of embryo microinjection in situ on the host plant and obtain high frequency mutagenesis, in excess of 55%, of the cinnabar and white eye pigmentation loci. Through pair matings, we obtained 100% transmission of w and cn alleles to the G3 generation and also established that both genes are located on autosomes. Our analysis of wing phenotype revealed an unexpected discovery of the participation of pteridine pigments in wing and wing-vein coloration, indicating a role for these pigments beyond eye color. We used amplicon sequencing to examine the extent of off-target mutagenesis in adults arising from injected eggs, which was found to be negligible or non-existent. Our data show that GWSS can be easily developed as a genetic model system for the Hemiptera, enabling the study of traits that contribute to the success of invasive pests and vectors of plant pathogens. This will facilitate novel genetic control strategies.}, }
@article {pmid35440579, year = {2022}, author = {de Rooij, MFM and Thus, YJ and Swier, N and Beijersbergen, RL and Pals, ST and Spaargaren, M}, title = {A loss-of-adhesion CRISPR-Cas9 screening platform to identify cell adhesion-regulatory proteins and signaling pathways.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {2136}, pmid = {35440579}, issn = {2041-1723}, support = {12539//KWF Kankerbestrijding (Dutch Cancer Society)/ ; 7873//KWF Kankerbestrijding (Dutch Cancer Society)/ ; 10275//KWF Kankerbestrijding (Dutch Cancer Society)/ ; }, mesh = {Agammaglobulinaemia Tyrosine Kinase/genetics/metabolism ; *CRISPR-Cas Systems ; Cell Adhesion/genetics ; Humans ; Integrins/metabolism ; *Leukemia/drug therapy ; Protein Kinase Inhibitors/therapeutic use ; Signal Transduction ; Tumor Microenvironment ; }, abstract = {The clinical introduction of the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, which targets B-cell antigen-receptor (BCR)-controlled integrin-mediated retention of malignant B cells in their growth-supportive lymphoid organ microenvironment, provided a major breakthrough in lymphoma and leukemia treatment. Unfortunately, a significant subset of patients is intrinsically resistant or acquires resistance against ibrutinib. Here, to discover novel therapeutic targets, we present an unbiased loss-of-adhesion CRISPR-Cas9 knockout screening method to identify proteins involved in BCR-controlled integrin-mediated adhesion. Illustrating the validity of our approach, several kinases with an established role in BCR-controlled adhesion, including BTK and PI3K, both targets for clinically applied inhibitors, are among the top hits of our screen. We anticipate that pharmacological inhibitors of the identified targets, e.g. PAK2 and PTK2B/PYK2, may have great clinical potential as therapy for lymphoma and leukemia patients. Furthermore, this screening platform is highly flexible and can be easily adapted to identify cell adhesion-regulatory proteins and signaling pathways for other stimuli, adhesion molecules, and cell types.}, }
@article {pmid35440051, year = {2022}, author = {Tao, R and Wang, Y and Hu, Y and Jiao, Y and Zhou, L and Jiang, L and Li, L and He, X and Li, M and Yu, Y and Chen, Q and Yao, S}, title = {WT-PE: Prime editing with nuclease wild-type Cas9 enables versatile large-scale genome editing.}, journal = {Signal transduction and targeted therapy}, volume = {7}, number = {1}, pages = {108}, pmid = {35440051}, issn = {2059-3635}, support = {No. U19A2002//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81771220//National Natural Science Foundation of China (National Science Foundation of China)/ ; No.81974238//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81974238//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. U19A2002//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81771220//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81771220//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. U19A2002//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81771220//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81974238//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81771220//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. U19A2002//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. U19A2002//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81974238//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81771220//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. U19A2002//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81974238//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81771220//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81771220//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. U19A2002//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81974238//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81974238//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81771220//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. U19A2002//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81771220//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. U19A2002//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81974238//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81771220//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81974238//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. U19A2002//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81974238//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81771220//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. U19A2002//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81974238//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. 81771220//National Natural Science Foundation of China (National Science Foundation of China)/ ; No. U19A2002//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; RNA, Guide/genetics ; RNA-Directed DNA Polymerase/genetics ; }, abstract = {Large scale genomic aberrations including duplication, deletion, translocation, and other structural changes are the cause of a subtype of hereditary genetic disorders and contribute to onset or progress of cancer. The current prime editor, PE2, consisting of Cas9-nickase and reverse transcriptase enables efficient editing of genomic deletion and insertion, however, at small scale. Here, we designed a novel prime editor by fusing reverse transcriptase (RT) to nuclease wild-type Cas9 (WT-PE) to edit large genomic fragment. WT-PE system simultaneously introduced a double strand break (DSB) and a single 3' extended flap in the target site. Coupled with paired prime editing guide RNAs (pegRNAs) that have complementary sequences in their 3' terminus while target different genomic regions, WT-PE produced bi-directional prime editing, which enabled efficient and versatile large-scale genome editing, including large fragment deletion up to 16.8 megabase (Mb) pairs and chromosomal translocation. Therefore, our WT-PE system has great potential to model or treat diseases related to large-fragment aberrations.}, }
@article {pmid35438517, year = {2022}, author = {Sherkow, JS}, title = {Immaculate Conception? Priority and Invention in the CRISPR Patent Dispute.}, journal = {The CRISPR journal}, volume = {5}, number = {2}, pages = {174-180}, doi = {10.1089/crispr.2022.0033}, pmid = {35438517}, issn = {2573-1602}, mesh = {*CRISPR-Cas Systems/genetics ; Dissent and Disputes ; *Gene Editing ; Inventions ; RNA, Guide/genetics ; }, abstract = {The U.S. Patent Trial and Appeal Board (PTAB), in an interference proceeding decided in February 2022, concluded that researchers at the Broad Institute (Cambridge, MA) were the first to "conceive" of using single-guide RNA CRISPR-Cas9 genome editing in eukaryotic cells in 2012. The PTAB reached this verdict even though competing researchers at the University of California, Berkeley, among other institutions, could document the idea 7 months earlier. Understanding the basis for the PTAB's decision turns on patent law's particular "conception" requirement. In this study, I explain that requirement, detail the PTAB's interference decision, and discuss the decision's practical effects on CRISPR technology and routine science.}, }
@article {pmid35438515, year = {2022}, author = {Zuo, Z and Babu, K and Ganguly, C and Zolekar, A and Newsom, S and Rajan, R and Wang, YC and Liu, J}, title = {Rational Engineering of CRISPR-Cas9 Nuclease to Attenuate Position-Dependent Off-Target Effects.}, journal = {The CRISPR journal}, volume = {5}, number = {2}, pages = {329-340}, doi = {10.1089/crispr.2021.0076}, pmid = {35438515}, issn = {2573-1602}, support = {R15 HL147265/HL/NHLBI NIH HHS/United States ; P20 GM103640/GM/NIGMS NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems/genetics ; DNA Cleavage ; Endonucleases/genetics ; *Gene Editing ; Humans ; RNA/chemistry ; }, abstract = {The RNA-guided Cas9 nuclease from Streptococcus pyogenes has become an important gene-editing tool. However, its intrinsic off-target activity is a major challenge for biomedical applications. Distinct from some reported engineering strategies that specifically target a single domain, we rationally introduced multiple amino acid substitutions across multiple domains in the enzyme to create potential high-fidelity variants, considering the Cas9 specificity is synergistically determined by various domains. We also exploited our previously derived atomic model of activated Cas9 complex structure for guiding new modifications. This approach has led to the identification of the HSC1.2 Cas9 variant with enhanced specificity for DNA cleavage. While the enhanced specificity associated with the HSC1.2 variant appeared to be position-dependent in the in vitro cleavage assays, the frequency of off-target DNA editing with this Cas9 variant is much less than that of the wild-type Cas9 in human cells. The potential mechanisms causing the observed position-dependent effect were investigated through molecular dynamics simulation. Our discoveries establish a solid foundation for leveraging structural and dynamic information to develop Cas9-like enzymes with high specificity in gene editing.}, }
@article {pmid35438514, year = {2022}, author = {Sun, W and Wang, Y}, title = {SuperFi-Cas9: High Fidelity Meets High Activity.}, journal = {The CRISPR journal}, volume = {5}, number = {2}, pages = {171-173}, doi = {10.1089/crispr.2022.29146.ywa}, pmid = {35438514}, issn = {2573-1602}, mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; RNA, Guide ; }, }
@article {pmid35438513, year = {2022}, author = {Barrangou, R}, title = {CRISPR à la carte.}, journal = {The CRISPR journal}, volume = {5}, number = {2}, pages = {170}, doi = {10.1089/crispr.2022.29147.rba}, pmid = {35438513}, issn = {2573-1602}, mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; }, }
@article {pmid35437598, year = {2022}, author = {Yagita, Y and Abe, Y and Fujiki, Y}, title = {De novo formation and maintenance of mammalian peroxisomes in cultured PEX16-knockout cells generated by CRISPR/Cas9.}, journal = {Journal of cell science}, volume = {135}, number = {9}, pages = {}, doi = {10.1242/jcs.258377}, pmid = {35437598}, issn = {1477-9137}, support = {JP26116007//Japan Society for the Promotion of Science/ ; //Takeda Science Foundation/ ; //Naito Foundation/ ; //Japan Foundation for Applied Enzymology/ ; //Novartis Foundation (Japan) for the Promotion of Science/ ; JP19K07386//Ministry of Education, Culture, Sports, Science, and Technology/ ; JP19K07386//Japan Society for the Promotion of Science/ ; //Novartis Foundation/ ; }, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Cell Line ; Humans ; Intracellular Membranes/metabolism ; Mammals/metabolism ; Membrane Proteins/metabolism ; *Peroxisomes/metabolism ; }, abstract = {Mammalian PEX16 has been considered essential for generating and maintaining peroxisomal membranes. This view is based primarily on the finding that fibroblasts from several PEX16-deficient patients are devoid of peroxisomal structures but can form peroxisomes upon expression of PEX16. However, unlike these patient-derived cells, pex16 mutants in other model organisms contain partially functional peroxisomes. Here, we report that PEX16-knockout (KO) cells derived from three mammalian cultured cell lines comprise cells containing a fewer number of enlarged peroxisomes and cells lacking peroxisomes. We also suggest that PEX16 accelerates the process by which peroxisome-less cells form peroxisomal membranes and subsequently establish mature peroxisomes, independently of its ability to mediate peroxisomal targeting of PEX3. Nevertheless, PEX16 is not absolutely required for this process. Moreover, a well-known patient-derived PEX16 mutant inhibits the de novo formation of peroxisomal membranes. Our findings suggest that although PEX16 is undoubtedly important for optimal peroxisomal membrane biogenesis, mammalian cells may be able to form peroxisomes de novo and maintain the organelles without the aid of PEX16.}, }
@article {pmid35435904, year = {2022}, author = {Hu, C and Doerksen, T and Bugbee, T and Wallace, NA and Palinski, R}, title = {Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {181}, pages = {}, doi = {10.3791/62583}, pmid = {35435904}, issn = {1940-087X}, support = {P20 GM130448/GM/NIGMS NIH HHS/United States ; R15 CA242057/CA/NCI NIH HHS/United States ; }, mesh = {CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; *DNA Repair/genetics ; High-Throughput Nucleotide Sequencing ; Mutation ; }, abstract = {Double strand breaks (DSBs) in DNA are the most cytotoxic type of DNA damage. Because a myriad of insults can result in these lesions (e.g., replication stress, ionizing radiation, unrepaired UV damage), DSBs occur in most cells each day. In addition to cell death, unrepaired DSBs reduce genome integrity and the resulting mutations can drive tumorigenesis. These risks and the prevalence of DSBs motivate investigations into the mechanisms by which cells repair these lesions. Next generation sequencing can be paired with the induction of DSBs by ionizing radiation to provide a powerful tool to precisely define the mutations associated with DSB repair defects. However, this approach requires computationally challenging and cost prohibitive whole genome sequencing to detect the repair of the randomly occurring DSBs associated with ionizing radiation. Rare cutting endonucleases, such as I-Sce1, provide the ability to generate a single DSB, but their recognition sites must be inserted into the genome of interest. As a result, the site of repair is inherently artificial. Recent advances allow guide RNA (sgRNA) to direct a Cas9 endonuclease to any genome locus of interest. This could be applied to the study of DSB repair making next generation sequencing more cost effective by allowing it to be focused on the DNA flanking the Cas9-induced DSB. The goal of the manuscript is to demonstrate the feasibility of this approach by presenting a protocol that can define mutations that stem from the repair of a DSB upstream of the CD4 gene. The protocol can be adapted to determine changes in the mutagenic potential of DSB associated with exogenous factors, such as repair inhibitors, viral protein expression, mutations, and environmental exposures with relatively limited computation requirements. Once an organism's genome has been sequenced, this method can be theoretically employed at any genomic locus and in any cell culture model of that organism that can be transfected. Similar adaptations of the approach could allow comparisons of repair fidelity between different loci in the same genetic background.}, }
@article {pmid35435766, year = {2022}, author = {Vaca, DJ and Thibau, A and Leisegang, MS and Malmström, J and Linke, D and Eble, JA and Ballhorn, W and Schaller, M and Happonen, L and Kempf, VAJ}, title = {Interaction of Bartonella henselae with Fibronectin Represents the Molecular Basis for Adhesion to Host Cells.}, journal = {Microbiology spectrum}, volume = {}, number = {}, pages = {e0059822}, doi = {10.1128/spectrum.00598-22}, pmid = {35435766}, issn = {2165-0497}, abstract = {Bacterial adhesion to the host is the most decisive step in infections. Trimeric autotransporter adhesins (TAA) are important pathogenicity factors of Gram-negative bacteria. The prototypic TAA Bartonella adhesin A (BadA) from human-pathogenic Bartonella henselae mediates bacterial adherence to endothelial cells (ECs) and extracellular matrix proteins. Here, we determined the interaction between BadA and fibronectin (Fn) to be essential for bacterial host cell adhesion. BadA interactions occur within the heparin-binding domains of Fn. The exact binding sites were revealed by mass spectrometry analysis of chemically cross-linked whole-cell bacteria and Fn. Specific BadA interactions with defined Fn regions represent the molecular basis for bacterial adhesion to ECs and these data were confirmed by BadA-deficient bacteria and CRISPR-Cas knockout Fn host cells. Interactions between TAAs and the extracellular matrix might represent the key step for adherence of human-pathogenic Gram-negative bacteria to the host. IMPORTANCE Deciphering the mechanisms of bacterial host cell adhesion is a clue for preventing infections. We describe the underestimated role that the extracellular matrix protein fibronectin plays in the adhesion of human-pathogenic Bartonella henselae to host cells. Fibronectin-binding is mediated by a trimeric autotransporter adhesin (TAA) also present in many other human-pathogenic Gram-negative bacteria. We demonstrate that both TAA and host-fibronectin contribute significantly to bacterial adhesion, and we present the exact sequence of interacting amino acids from both proteins. Our work shows the domain-specific pattern of interaction between the TAA and fibronectin to adhere to host cells and opens the perspective to fight bacterial infections by inhibiting bacterial adhesion which represents generally the first step in infections.}, }
@article {pmid35433512, year = {2022}, author = {Lu, P and Chen, J and Li, Z and Li, Z and Zhang, J and Kan, B and Pang, B}, title = {Visual Identification and Serotyping of Toxigenic Vibrio cholerae Serogroups O1 and O139 With CARID.}, journal = {Frontiers in cellular and infection microbiology}, volume = {12}, number = {}, pages = {863435}, pmid = {35433512}, issn = {2235-2988}, mesh = {*Cholera/diagnosis/microbiology ; Cholera Toxin ; Humans ; Serogroup ; Serotyping ; *Vibrio cholerae O1/genetics ; }, abstract = {There is a growing demand for rapid, sensitive, field-deployable nucleic acid tests for cholera, which usually occurs in rural areas. In this study, we developed a Cas12a-assisted rapid isothermal detection (CARID) system for the detection of toxigenic V. cholerae serogroups O1 and O139 by combining recombinase-aided amplification and CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins). The results can be determined by fluorescence signal and visualized by lateral flow dipstick. We identified 154 V. cholerae strains and 129 strains of other intestinal diarrheagenic bacteria with a 100% coincidence rate. The limit of detection of CARID was 20 copies/reaction of V. cholerae genomic DNA, which is comparable to that of polymerase chain reaction (PCR) and qPCR. Multiple-CARID was also established for efficiency and economic considerations with an acceptable decrease in sensitivity. Simulated sample tests showed that CARID is suitable for complex samples. In conclusion, CARID is a rapid, sensitive, economically efficient, and portable method for the detection of V. cholerae, which makes it suitable for field responses to cholera.}, }
@article {pmid35432328, year = {2022}, author = {Moraes, L and Trentini, MM and Fousteris, D and Eto, SF and Chudzinski-Tavassi, AM and Leite, LCC and Kanno, AI}, title = {CRISPR/Cas9 Approach to Generate an Auxotrophic BCG Strain for Unmarked Expression of LTAK63 Adjuvant: A Tuberculosis Vaccine Candidate.}, journal = {Frontiers in immunology}, volume = {13}, number = {}, pages = {867195}, pmid = {35432328}, issn = {1664-3224}, mesh = {Adjuvants, Immunologic ; Adjuvants, Pharmaceutic ; Animals ; BCG Vaccine/genetics ; CRISPR-Cas Systems ; Escherichia coli ; Mice ; *Tuberculosis ; *Tuberculosis Vaccines/genetics ; }, abstract = {Tuberculosis is one of the deadliest infectious diseases and a huge healthcare burden in many countries. New vaccines, including recombinant BCG-based candidates, are currently under evaluation in clinical trials. Our group previously showed that a recombinant BCG expressing LTAK63 (rBCG-LTAK63), a genetically detoxified subunit A of heat-labile toxin (LT) from Escherichia coli, induces improved protection against Mycobacterium tuberculosis (Mtb) in mouse models. This construct uses a traditional antibiotic resistance marker to enable heterologous expression. In order to avoid the use of these markers, not appropriate for human vaccines, we used CRISPR/Cas9 to generate unmarked mutations in the lysA gene, thus obtaining a lysine auxotrophic BCG strain. A mycobacterial vector carrying lysA and ltak63 gene was used to complement the auxotrophic BCG which co-expressed the LTAK63 antigen (rBCGΔ-LTAK63) at comparable levels to the original construct. The intranasal challenge with Mtb confirmed the superior protection induced by rBCGΔ-LTAK63 compared to wild-type BCG. Furthermore, mice immunized with rBCGΔ-LTAK63 showed improved lung function. In this work we showed the practical application of CRISPR/Cas9 in the tuberculosis vaccine development field.}, }
@article {pmid35430708, year = {2022}, author = {Bhoria, S and Yadav, J and Yadav, H and Chaudhary, D and Jaiwal, R and Jaiwal, PK}, title = {Current advances and future prospects in production of recombinant insulin and other proteins to treat diabetes mellitus.}, journal = {Biotechnology letters}, volume = {}, number = {}, pages = {}, pmid = {35430708}, issn = {1573-6776}, abstract = {Diabetes mellitus is the most prevalent deadly disease caused by the destruction and dysfunction of pancreatic β cells that consequentially increased blood glucose levels. The management of this disease via external administration of insulin/insulin analogs has been difficult and challenging due to their limited production and accessibility at affordable prices. The conventional insulin production platforms (Escherichia coli, Saccharomyces cerevisiae and mammalian cell lines) with limited scalability and high upstream process costs have not been successful in meeting the rapidly increasing insulin demands. However, plants have been used as safe, scalable, environmentally friendly and cost-effective high capacity production platforms for recombinant orally delivered insulin. Recent technological advances in genome engineering and editing technologies for adequate insulin and insulin analogs production, renewable cellular sources of insulin through transplantation of islets or insulin-producing cells and reprogramming or differentiation of non β cells into β-like cells, used either alone or in combination, for diabetes containment are reviewed here along with their future prospects.}, }
@article {pmid35430408, year = {2022}, author = {Moon, J and Song, J and Jang, H and Kang, H and Huh, YM and Son, HY and Rho, HW and Park, M and Talwar, CS and Park, KH and Woo, E and Lim, J and Lim, EK and Jung, J and Jung, Y and Park, HG and Kang, T}, title = {Ligation-free isothermal nucleic acid amplification.}, journal = {Biosensors &amp; bioelectronics}, volume = {209}, number = {}, pages = {114256}, doi = {10.1016/j.bios.2022.114256}, pmid = {35430408}, issn = {1873-4235}, mesh = {Animals ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; DNA/genetics ; Mice ; Nucleic Acid Amplification Techniques/methods ; RNA ; RNA, Messenger ; }, abstract = {In this study, we uncover a ligation-free DNA extension method in two adjacent fragmented probes, which are hybridized to target RNA, for developing a ligation-free nucleic acid amplification reaction. In this reaction, DNA elongation occurs from a forward probe to a phosphorothioated-hairpin probe in the presence of target RNA regardless of ligation. The second DNA elongation then occurs simultaneously at the nick site of the phosphorothioated probe and the self-priming region. Therefore, the binding site of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 12a is repeatedly amplified, inducing a fluorescence signal in the presence of CRISPR-Cas12a. This ligation-free isothermal gene amplification method enables the detection of target RNA with 49.2 fM sensitivity. Moreover, two types of mRNA detection are feasible, thus, demonstrating the potential of this method for cancer companion diagnostics. Notably, the proposed method also demonstrates efficacy when applied for the detection of mRNA extracted from human cells and tumor-bearing mouse tissue and urine samples. Hence, this newly developed ligation-free isothermal nucleic acid amplification system is expected to be widely used in a variety of gene detection platforms.}, }
@article {pmid35429769, year = {2022}, author = {Pan, Y and Luan, X and Zeng, F and Xu, Q and Li, Z and Gao, Y and Liu, X and Li, X and Han, X and Shen, J and Song, Y}, title = {Hollow covalent organic framework-sheltering CRISPR/Cas12a as an in-vivo nanosensor for ATP imaging.}, journal = {Biosensors &amp; bioelectronics}, volume = {209}, number = {}, pages = {114239}, doi = {10.1016/j.bios.2022.114239}, pmid = {35429769}, issn = {1873-4235}, mesh = {Adenosine Triphosphate ; Animals ; *Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; DNA, Single-Stranded ; Gene Editing/methods ; *Metal-Organic Frameworks ; Oligonucleotides ; }, abstract = {In addition to applications in genome editing, clustered regularly interspaced short palindromic repeats (CRISPR) have recently been engineered for medical diagnostics based on their trans-cleavage activity owing to their high base resolution and isothermal signal amplification. However, trans-cleavage activity is too fragile to be applied in vivo. Herein, we introduce a hollow covalent organic framework (COF)-sheltering CRISPR/aptamer-based sensor (h-CCS) for ATP imaging in living animals. The CRISPR/aptamer-based complex is comprised of the CRISPR-Cas12a system, fluorophore quencher-labeled single-stranded DNA substrate (ssDNA-FQ), and a DNA activator that pre-hybridizes with ATP aptamer to prevent the trans-cleavage activity of the Cas12a system in the absence of ATP. After being encapsulated in a hollow COF, the constructed nanoreactor is highly robust and can be lit up by ATP for in vivo imaging. Considering the unique properties of h-CCS, this strategy offers great potential to broaden applications of not only CRISPR-Cas systems but also other proteins in porous matrixes for clinical diagnostics, medical research, and biomimetic nanodevices.}, }
@article {pmid35429445, year = {2022}, author = {Chen, Q and Wen, Y}, title = {Target recognition triggered CRISPR-Cas12a assisted allosteric scaffold for sensitively analyzing bacterial infection after dental implantation.}, journal = {Analytical biochemistry}, volume = {647}, number = {}, pages = {114666}, doi = {10.1016/j.ab.2022.114666}, pmid = {35429445}, issn = {1096-0309}, mesh = {*Bacterial Infections/diagnosis/genetics ; *CRISPR-Cas Systems ; DNA ; Dental Implantation ; Humans ; Limit of Detection ; }, abstract = {Accurate identification and sensitive quantification of infected bacteria after dental implant both play crucial roles in early-diagnosis of bacterial infection and guiding medicine applications. Herein, we propose a sensitive and accurate bacteria detection method based on CRISPR-Cas12a system assisted allosteric scaffold. In the method, the allosteric scaffold takes the responsibility of specifically identifying target bacteria and inducing CRISPR-Cas12a based signal amplification. Eventually, the method exhibits a wide detection range from 6 × 106 cfu/mL to 6 × 102 cfu/mL with the limit of detection (LOD) of 47 cfu/mL. Furthermore, the established approach also possesses a high specificity due to high selectivity of aptamer and robust accuracy in recognizing double strand DNA by CRISPR-Cas12a system. We believe that this work can provide new strategies in the field of diagnosing bacterial infections after dental implantation.}, }
@article {pmid35427904, year = {2022}, author = {Lee, HM and Kim, AH and Hwang, S and Jung, J and Seol, H and Sung, JJ and Jeong, SM and Choi, YM and Jun, JK and Kim, HS and Jang, J}, title = {Generation of αMHC-EGFP knock-in in human pluripotent stem cell line, SNUe003-A-3 using CRISPR/Cas9-based gene targeting.}, journal = {Stem cell research}, volume = {61}, number = {}, pages = {102779}, doi = {10.1016/j.scr.2022.102779}, pmid = {35427904}, issn = {1876-7753}, mesh = {*CRISPR-Cas Systems/genetics ; Cell Line ; Gene Targeting ; Genes, Reporter ; Green Fluorescent Proteins/genetics/metabolism ; Humans ; Myosin Heavy Chains/genetics ; *Pluripotent Stem Cells/metabolism ; }, abstract = {The cardiac muscle-specific protein, α-myosin heavy chain (αMHC), is a major component of cardiac muscle filaments involved in cardiac muscle contraction. Here, we established an αMHC-enhanced fluorescent protein (EGFP) knock-in human pluripotent stem cell (hPSC) line by linking the EGFP gene to the C-terminal region of αMHC via a 2A non-joining peptide using CRISPR/Cas9 nuclease. The EGFP reporter precisely reflected the endogenous level of αMHC upon the induction of cardiac differentiation. This reporter cell line will be a valuable platform for cardiotoxicity tests, drug screening, and investigating the pathological mechanisms of cardiomyocytes.}, }
@article {pmid35427438, year = {2022}, author = {Kelkar, A and Groth, T and Neelamegham, S}, title = {Forward Genetic Screens of Human Glycosylation Pathways Using the GlycoGene CRISPR Library.}, journal = {Current protocols}, volume = {2}, number = {4}, pages = {e402}, doi = {10.1002/cpz1.402}, pmid = {35427438}, issn = {2691-1299}, support = {HL103411/GF/NIH HHS/United States ; GM133195/GF/NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems/genetics ; Gene Library ; *Genetic Testing ; Glycosylation ; Humans ; Lectins/genetics ; }, abstract = {CRISPR-Cas9-based forward genetic screens represent a powerful discovery platform to uncover genes regulating specific biological processes. This article describes a method for utilizing a freely available GlycoGene CRISPR library to knock out any gene participating in human glycosylation in arbitrary cell types. The end product is a stable GlycoGene CRISPR knockout cell library, where each cell contains one or more sgRNA and lacks corresponding function. The cell library can be screened using various lectin/antibody reagents. It can also be applied in functional assays to establish glycan structure-glycogene-glycopathway relationships. This is a powerful systems glycobiology strategy for dissecting glycosylation pathways and processes. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Scale-up and NGS validation of the GlycoGene CRISPR plasmid library Basic Protocol 2: Preparation of a GlycoGene CRISPR lentivirus pool and an isogenic cell line stably expressing Cas9 nuclease Basic Protocol 3: Preparation of a GlycoGene CRISPR cell library, self-inactivation of Cas9, and library validation by NGS Basic Protocol 4: Enrichment of lectin-binding or non-binding cells and related multiplex NGS data acquisition Basic Protocol 5: Bioinformatics pathway analysis.}, }
@article {pmid35426306, year = {2022}, author = {Gao, ZF and Zheng, LL and Dong, LM and Li, JZ and Shen, Y and Chen, P and Xia, F}, title = {Label-Free Resonance Rayleigh Scattering Amplification for Lipopolysaccharide Detection and Logical Circuit by CRISPR/Cas12a-Driven Guanine Nanowire Assisted Non-Cross-Linking Hybridization Chain Reaction.}, journal = {Analytical chemistry}, volume = {94}, number = {16}, pages = {6371-6379}, doi = {10.1021/acs.analchem.2c00848}, pmid = {35426306}, issn = {1520-6882}, mesh = {CRISPR-Cas Systems/genetics ; Guanine ; Lipopolysaccharides ; Logic ; *Nanowires ; }, abstract = {Although the CRISPR/Cas system has pioneered a new generation of analytical techniques, there remain many challenges in developing a label-free, accurate, and reliable CRISPR/Cas-based assay for reporting the levels of low abundance biomolecules in complex biological samples. Here, we reported a novel CRISPR-derived resonance Rayleigh scattering (RRS) amplification strategy and logical circuit based on a guanine nanowire (G-wire) assisted non-cross-linking hybridization chain reaction (GWancHCR) for label-free detection of lipopolysaccharide (LPS). In the presence of a target, the protospacer-adjacent motif-inserted aptamer is rationally designed to specifically combine with LPS rather than Cas12a, suppressing the trans-cleavage activity of CRISPR/Cas12a and retaining the reporter probes to trigger non-cross-linking aggregation. Owing to the automatic hybridization chain reaction (HCR), in the presence of Mg2+, the released G-quadruplex sequence aggregated to assemble the G-wire superstructure through non-cross-linking. As a result, a dramatically amplified RRS intensity is observed, allowing for reporting LPS levels in a low detection limit of 0.17 pg/mL and a wide linear range among 1.0-100.0 ng/mL. Moreover, this reaction event is capable of programming to perform classical Boolean logic tree analysis, including basic logic computing and complex integrated logic circuits. This study comprehensively analyzed with respect to information flow, matter (molecular events), and energy (RRS), revealing the potential promise in designing of molecular-level "Internet of Things", intelligent computing, and sensing systems.}, }
@article {pmid35422794, year = {2022}, author = {Kamruzzaman, M and Yan, A and Castro-Escarpulli, G}, title = {Editorial: CRISPR-Cas Systems in Bacteria and Archaea.}, journal = {Frontiers in microbiology}, volume = {13}, number = {}, pages = {887778}, pmid = {35422794}, issn = {1664-302X}, }
@article {pmid35422517, year = {2022}, author = {Sinan, S and Russell, R}, title = {A tweak and a peek: How Cas9 pries open double-stranded DNA to check its sequence.}, journal = {Nature structural &amp; molecular biology}, volume = {29}, number = {4}, pages = {286-288}, pmid = {35422517}, issn = {1545-9985}, mesh = {*CRISPR-Cas Systems ; *DNA ; }, }
@article {pmid35422516, year = {2022}, author = {Cofsky, JC and Soczek, KM and Knott, GJ and Nogales, E and Doudna, JA}, title = {CRISPR-Cas9 bends and twists DNA to read its sequence.}, journal = {Nature structural &amp; molecular biology}, volume = {29}, number = {4}, pages = {395-402}, pmid = {35422516}, issn = {1545-9985}, support = {U01 AI142817/AI/NIAID NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems/genetics ; DNA/metabolism ; Endonucleases/metabolism ; Gene Editing ; *RNA, Guide/metabolism ; }, abstract = {In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM). Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism. Here we show that Cas9 sharply bends and undertwists DNA on PAM binding, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryogenic-electron microscopy (cryo-EM) structures of Cas9-RNA-DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 'reads' snippets of DNA to locate target sites within a vast excess of nontarget DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency.}, }
@article {pmid35422284, year = {2022}, author = {Li, Q and Lv, X and Tang, C and Yin, C}, title = {Co-delivery of doxorubicin and CRISPR/Cas9 or RNAi-expressing plasmid by chitosan-based nanoparticle for cancer therapy.}, journal = {Carbohydrate polymers}, volume = {287}, number = {}, pages = {119315}, doi = {10.1016/j.carbpol.2022.119315}, pmid = {35422284}, issn = {1879-1344}, mesh = {CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; *Chitosan ; Doxorubicin/pharmacology ; Drug Carriers ; Drug Delivery Systems/methods ; Folic Acid ; *Frontotemporal Dementia ; Humans ; *Nanoparticles ; *Neoplasms/drug therapy/genetics ; Plasmids/genetics ; RNA Interference ; Survivin/genetics/metabolism ; }, abstract = {Folic acid (FA) and 2-(Diisopropylamino) ethyl methacrylate (DPA) double grafted trimethyl chitosan (TMC) nanoparticles (FTD NPs) were synthesized for the co-delivery of doxorubicin (DOX) and Survivin CRISPR/Cas9-expressing plasmid (sgSurvivin pDNA) or Survivin shRNA-expressing plasmid (iSur pDNA). FA modification enhanced the uptake of DOX and pDNA loaded into FTD NPs in tumor cells. A rapid release of DOX was triggered under acidic conditions due to pH-sensitiveness of FTD NPs arising from DPA conjugation. Negligible differences between FTD/sgSurvivin pDNA NPs and FTD/iSur pDNA NPs demonstrated that RNA interference (RNAi) and CRISPR/Cas9 technologies possessed comparable antitumor efficiency. Notably, the in vitro and in vivo antitumor efficacies of FTD/DOX/sgSurvivin pDNA NPs were superior to those of single delivery of DOX or sgSurvivin pDNA, while were comparable to those of FTD/DOX/iSur pDNA NPs. These results suggested that the combination of chemotherapeutics and CRISPR/Cas9 systems would provide a potential modality for cancer therapy.}, }
@article {pmid35421847, year = {2022}, author = {Zhang, F and Meier, AB and Sinnecker, D and Engelhardt, S and Lipp, P and Laugwitz, KL and Dorn, T and Moretti, A}, title = {Generation of heterozygous (MRli003-A-5) and homozygous (MRli003-A-6) voltage-sensing knock-in human iPSC lines by CRISPR/Cas9 editing of the AAVS1 locus.}, journal = {Stem cell research}, volume = {61}, number = {}, pages = {102785}, doi = {10.1016/j.scr.2022.102785}, pmid = {35421847}, issn = {1876-7753}, support = {788381/ERC_/European Research Council/International ; }, mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Homozygote ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Myocytes, Cardiac/metabolism ; }, abstract = {Assessment of the electrophysiological properties of cardiomyocytes is necessary for phenotyping cardiac disorders and for drug screening. Optical action potential imaging using a genetically encoded voltage-sensing fluorescent protein (VSFP) allows for high-throughput functional characterization of cardiomyocytes, which offers an advantage over the traditional patch-clamp technique. Here, we knocked VSFP into the AAVS1 safe harbor locus of human iPSCs, generating two stable voltage indicator lines - one heterozygous (MRIi003-A-5) and the other homozygous (MRI003-A-6). Both lines can be used for optical membrane potential recordings and provide a powerful platform for a wide range of applications in cardiovascular biomedicine.}, }
@article {pmid35421656, year = {2022}, author = {Fu, R and Wang, Y and Liu, Y and Liu, H and Zhao, Q and Zhang, Y and Wang, C and Li, Z and Jiao, B and He, Y}, title = {CRISPR-Cas12a based fluorescence assay for organophosphorus pesticides in agricultural products.}, journal = {Food chemistry}, volume = {387}, number = {}, pages = {132919}, doi = {10.1016/j.foodchem.2022.132919}, pmid = {35421656}, issn = {1873-7072}, mesh = {Acetylcholinesterase/genetics ; *Biosensing Techniques ; CRISPR-Cas Systems ; *DNA, Catalytic ; Manganese Compounds ; Organophosphorus Compounds ; Oxides ; *Pesticides ; }, abstract = {Herein, we propose a sensitive fluorescent assay for organophosphorus pesticides (OPs) detection based on a novel strategy of activating the CRISPR-Cas12a system. Specifically, acetylcholinesterase (AChE) hydrolyzes acetylthiocholine into thiocholine (TCh). Subsequently, TCh induces the degradation of MnO2 nanosheets and generates sufficient Mn2+ ions to activate the Mn2+-dependent DNAzyme. Then, as the catalytic product of activated DNAzyme, the short DNA strand activates the CRISPR-Cas12a system to cleave the fluorophore-quencher-labeled DNA reporter (FQ) probe effectively; thus, increasing the fluorescence intensity (FI) in the solution. However, in the presence of OPs, the activity of AChE is suppressed, resulting in a decrease in FI. Under optimized conditions, the limits of detection for paraoxon, dichlorvos, and demeton were 270, 406, and 218 pg/mL, respectively. Benefiting from the outstanding MnO2 nanosheets properties and three rounds of enzymatic signal amplification, the proposed fluorescence assay holds great potential for the detection of OPs in agricultural products.}, }
@article {pmid35421352, year = {2022}, author = {Bari, SMN and Chou-Zheng, L and Howell, O and Hossain, M and Hill, CM and Boyle, TA and Cater, K and Dandu, VS and Thomas, A and Aslan, B and Hatoum-Aslan, A}, title = {A unique mode of nucleic acid immunity performed by a multifunctional bacterial enzyme.}, journal = {Cell host &amp; microbe}, volume = {30}, number = {4}, pages = {570-582.e7}, doi = {10.1016/j.chom.2022.03.001}, pmid = {35421352}, issn = {1934-6069}, mesh = {Bacteria/genetics ; *Bacteriophages/genetics ; CRISPR-Cas Systems ; Multifunctional Enzymes/genetics ; *Nucleic Acids ; Staphylococcus Phages/genetics ; }, abstract = {The perpetual arms race between bacteria and their viruses (phages) has given rise to diverse immune systems, including restriction-modification and CRISPR-Cas, which sense and degrade phage-derived nucleic acids. These complex systems rely upon production and maintenance of multiple components to achieve antiphage defense. However, the prevalence and effectiveness of minimal, single-component systems that cleave DNA remain unknown. Here, we describe a unique mode of nucleic acid immunity mediated by a single enzyme with nuclease and helicase activities, herein referred to as Nhi (nuclease-helicase immunity). This enzyme provides robust protection against diverse staphylococcal phages and prevents phage DNA accumulation in cells stripped of all other known defenses. Our observations support a model in which Nhi targets and degrades phage-specific replication intermediates. Importantly, Nhi homologs are distributed in diverse bacteria and exhibit functional conservation, highlighting the versatility of such compact weapons as major players in antiphage defense.}, }
@article {pmid35421333, year = {2022}, author = {Huiting, E and Bondy-Denomy, J}, title = {A single bacterial enzyme i(NHI)bits phage DNA replication.}, journal = {Cell host &amp; microbe}, volume = {30}, number = {4}, pages = {417-419}, doi = {10.1016/j.chom.2022.03.025}, pmid = {35421333}, issn = {1934-6069}, mesh = {Bacteria/genetics ; *Bacteriophages/genetics ; CRISPR-Cas Systems ; DNA Helicases/genetics ; DNA Replication ; }, abstract = {In this issue of Cell Host &amp; Microbe, Nayeemul Bari et al. discover an anti-phage immune system in bacteria that uses a single enzyme to accomplish the challenging feat of detecting phage DNA and limiting its replication. Unlike CRISPR-Cas and restriction modification (R-M) systems, which use sequence motifs, nuclease-helicase immunity (Nhi) is proposed to target phage-specific replication intermediates.}, }
@article {pmid35420793, year = {2022}, author = {Wang, J and Skeens, E and Arantes, PR and Maschietto, F and Allen, B and Kyro, GW and Lisi, GP and Palermo, G and Batista, VS}, title = {Structural Basis for Reduced Dynamics of Three Engineered HNH Endonuclease Lys-to-Ala Mutants for the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Associated 9 (CRISPR/Cas9) Enzyme.}, journal = {Biochemistry}, volume = {61}, number = {9}, pages = {785-794}, pmid = {35420793}, issn = {1520-4995}, support = {R01 GM136815/GM/NIGMS NIH HHS/United States ; R01 GM141329/GM/NIGMS NIH HHS/United States ; }, mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/chemistry/genetics ; *Endonucleases/chemistry ; }, abstract = {Many bacteria possess type-II immunity against invading phages or plasmids known as the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9) system to detect and degrade the foreign DNA sequences. The Cas9 protein has two endonucleases responsible for double-strand breaks (the HNH domain for cleaving the target strand of DNA duplexes and RuvC domain for the nontarget strand, respectively) and a single-guide RNA-binding domain where the RNA and target DNA strands are base-paired. Three engineered single Lys-to-Ala HNH mutants (K810A, K848A, and K855A) exhibit an enhanced substrate specificity for cleavage of the target DNA strand. We report in this study that in the wild-type (wt) enzyme, D835, Y836, and D837 within the Y836-containing loop (comprising E827-D837) adjacent to the catalytic site have uncharacterizable broadened 1H15N nuclear magnetic resonance (NMR) features, whereas remaining residues in the loop have different extents of broadened NMR spectra. We find that this loop in the wt enzyme exhibits three distinct conformations over the duration of the molecular dynamics simulations, whereas the three Lys-to-Ala mutants retain only one conformation. The versatility of multiple alternate conformations of this loop in the wt enzyme could help to recruit noncognate DNA substrates into the HNH active site for cleavage, thereby reducing its substrate specificity relative to the three mutants. Our study provides further experimental and computational evidence that Lys-to-Ala substitutions reduce dynamics of proteins and thus increase their stability.}, }
@article {pmid35419381, year = {2022}, author = {Happi Mbakam, C and Lamothe, G and Tremblay, JP}, title = {Therapeutic Strategies for Dystrophin Replacement in Duchenne Muscular Dystrophy.}, journal = {Frontiers in medicine}, volume = {9}, number = {}, pages = {859930}, pmid = {35419381}, issn = {2296-858X}, abstract = {Duchenne muscular dystrophy (DMD) is an X-linked hereditary disease characterized by progressive muscle wasting due to modifications in the DMD gene (exon deletions, nonsense mutations, intra-exonic insertions or deletions, exon duplications, splice site defects, and deep intronic mutations) that result in a lack of functional dystrophin expression. Many therapeutic approaches have so far been attempted to induce dystrophin expression and improve the patient phenotype. In this manuscript, we describe the relevant updates for some therapeutic strategies for DMD aiming to restore dystrophin expression. We also present and analyze in vitro and in vivo ongoing experimental approaches to treat the disease.}, }
@article {pmid35418239, year = {2022}, author = {Orazi, G and Collins, AJ and Whitaker, RJ}, title = {Prediction of Prophages and Their Host Ranges in Pathogenic and Commensal Neisseria Species.}, journal = {mSystems}, volume = {}, number = {}, pages = {e0008322}, doi = {10.1128/msystems.00083-22}, pmid = {35418239}, issn = {2379-5077}, abstract = {The genus Neisseria includes two pathogenic species, N. gonorrhoeae and N. meningitidis, and numerous commensal species. Neisseria species frequently exchange DNA with one another, primarily via transformation and homologous recombination and via multiple types of mobile genetic elements (MGEs). Few Neisseria bacteriophages (phages) have been identified, and their impact on bacterial physiology is poorly understood. Furthermore, little is known about the range of species that Neisseria phages can infect. In this study, we used three virus prediction tools to scan 248 genomes of 21 different Neisseria species and identified 1,302 unique predicted prophages. Using comparative genomics, we found that many predictions are dissimilar from prophages and other MGEs previously described to infect Neisseria species. We also identified similar predicted prophages in genomes of different Neisseria species. Additionally, we examined CRISPR-Cas targeting of each Neisseria genome and predicted prophage. While CRISPR targeting of chromosomal DNA appears to be common among several Neisseria species, we found that 20% of the prophages we predicted are targeted significantly more than the rest of the bacterial genome in which they were identified (i.e., backbone). Furthermore, many predicted prophages are targeted by CRISPR spacers encoded by other species. We then used these results to infer additional host species of known Neisseria prophages and predictions that are highly targeted relative to the backbone. Together, our results suggest that we have identified novel Neisseria prophages, several of which may infect multiple Neisseria species. These findings have important implications for understanding horizontal gene transfer between members of this genus. IMPORTANCE Drug-resistant Neisseria gonorrhoeae is a major threat to human health. Commensal Neisseria species are thought to serve as reservoirs of antibiotic resistance and virulence genes for the pathogenic species N. gonorrhoeae and N. meningitidis. Therefore, it is important to understand both the diversity of mobile genetic elements (MGEs) that can mediate horizontal gene transfer within this genus and the breadth of species these MGEs can infect. In particular, few bacteriophages (phages) are known to infect Neisseria species. In this study, we identified a large number of candidate phages integrated in the genomes of commensal and pathogenic Neisseria species, many of which appear to be novel phages. Importantly, we discovered extensive interspecies targeting of predicted phages by Neisseria CRISPR-Cas systems, which may reflect their movement between different species. Uncovering the diversity and host range of phages is essential for understanding how they influence the evolution of their microbial hosts.}, }
@article {pmid35415942, year = {2022}, author = {Bao, YY and Zhong, JT and Shen, LF and Dai, LB and Zhou, SH and Fan, J and Yao, HT and Lu, ZJ}, title = {Effect of Glut-1 and HIF-1α double knockout by CRISPR/CAS9 on radiosensitivity in laryngeal carcinoma via the PI3K/Akt/mTOR pathway.}, journal = {Journal of cellular and molecular medicine}, volume = {26}, number = {10}, pages = {2881-2894}, doi = {10.1111/jcmm.17303}, pmid = {35415942}, issn = {1582-4934}, support = {81372903//National Natural Science Foundation of China/ ; 2016C33144//Science and Technology Department of Zhejiang Province, China/ ; 2016ZA127//Zhejiang TCM Science and technology program of Zhejiang provincial administration of traditional Chinese medicine/ ; }, mesh = {CRISPR-Cas Systems ; *Carcinoma ; Cell Line, Tumor ; Glucose ; Humans ; Hypoxia ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics/metabolism ; *Laryngeal Neoplasms/genetics/metabolism/radiotherapy ; Phosphatidylinositol 3-Kinases/genetics/metabolism ; Proto-Oncogene Proteins c-akt/genetics/metabolism ; Radiation Tolerance/genetics ; TOR Serine-Threonine Kinases/genetics/metabolism ; Wortmannin ; }, abstract = {Hypoxic resistance is the main obstacle to radiotherapy for laryngeal carcinoma. Our previous study indicated that hypoxia-inducible factor 1α (HIF-1α) and glucose transporter 1 (Glut-1) double knockout reduced tumour biological behaviour in laryngeal carcinoma cells. However, their radioresistance mechanism remains unclear. In this study, cell viability was determined by CCK8 assay. Glucose uptake capability was evaluated by measurement of 18 F-fluorodeoxyglucose radioactivity. A tumour xenograft model was established by subcutaneous injection of Tu212 cells. Tumour histopathology was determined by haematoxylin and eosin staining, immunohistochemical staining, and TUNEL assays. Signalling transduction was evaluated by Western blotting. We found that hypoxia induced radioresistance in Tu212 cells accompanied by increased glucose uptake capability and activation of the PI3K/Akt/mTOR pathway. Inhibition of PI3K/Akt/mTOR activity abolished hypoxia-induced radioresistance and glucose absorption. Mechanistic analysis revealed that hypoxia promoted higher expressions of HIF-1α and Glut-1. Moreover, the PI3K/Akt/mTOR pathway was a positive mediator of HIF-1α and/or Glut-1 in the presence of irradiation. HIF-1α and/or Glut-1 knockout significantly reduced cell viability, glucose uptake and PI3K/Akt/mTOR activity, all of which were induced by hypoxia in the presence of irradiation. In vivo analysis showed that knockout of HIF-1α and/or Glut-1 also inhibited tumour growth by promoting cell apoptosis, more robustly compared with the PI3K inhibitor wortmannin, particularly in tumours with knockout of both HIF-1α and Glut-1. HIF-1α and/or Glut-1 knockout also abrogated PI3K/Akt/mTOR signalling transduction in tumour tissues, in a manner similar to wortmannin. HIF-1α and/or Glut-1 knockout facilitated radiosensitivity in laryngeal carcinoma Tu212 cells by regulation of the PI3K/Akt/mTOR pathway.}, }
@article {pmid35414398, year = {2022}, author = {López-Valls, M and Escalona-Noguero, C and Rodríguez-Díaz, C and Pardo, D and Castellanos, M and Milán-Rois, P and Martínez-Garay, C and Coloma, R and Abreu, M and Cantón, R and Galán, JC and Miranda, R and Somoza, Á and Sot, B}, title = {CASCADE: Naked eye-detection of SARS-CoV-2 using Cas13a and gold nanoparticles.}, journal = {Analytica chimica acta}, volume = {1205}, number = {}, pages = {339749}, pmid = {35414398}, issn = {1873-4324}, mesh = {*COVID-19/diagnosis ; CRISPR-Cas Systems ; Gold ; Humans ; *Metal Nanoparticles ; Nucleic Acid Amplification Techniques/methods ; *Nucleic Acids ; Pandemics ; RNA, Viral/genetics ; SARS-CoV-2/genetics ; }, abstract = {The COVID-19 pandemic has brought to light the need for fast and sensitive detection methods to prevent the spread of pathogens. The scientific community is making a great effort to design new molecular detection methods suitable for fast point-of-care applications. In this regard, a variety of approaches have been developed or optimized, including isothermal amplification of viral nucleic acids, CRISPR-mediated target recognition, and read-out systems based on nanomaterials. Herein, we present CASCADE (CRISPR/CAS-based Colorimetric nucleic Acid DEtection), a sensing system for fast and specific naked-eye detection of SARS-CoV-2 RNA. In this approach, viral RNA is recognized by the LwaCas13a CRISPR protein, which activates its collateral RNase activity. Upon target recognition, Cas13a cleaves ssRNA oligonucleotides conjugated to gold nanoparticles (AuNPs), thus inducing their colloidal aggregation, which can be easily visualized. After an exhaustive optimization of functionalized AuNPs, CASCADE can detect picomolar concentrations of SARS-CoV-2 RNA. This sensitivity is further increased to low femtomolar (3 fM) and even attomolar (40 aM) ranges when CASCADE is coupled to RPA or NASBA isothermal nucleic acid amplification, respectively. We finally demonstrate that CASCADE succeeds in detecting SARS-CoV-2 in clinical samples from nasopharyngeal swabs. In conclusion, CASCADE is a fast and versatile RNA biosensor that can be coupled to different isothermal nucleic acid amplification methods for naked-eye diagnosis of infectious diseases.}, }
@article {pmid35414381, year = {2022}, author = {Sohail, M and Xie, S and Zhang, X and Li, B}, title = {Methodologies in visualizing the activation of CRISPR/Cas: The last mile in developing CRISPR-Based diagnostics and biosensing - A review.}, journal = {Analytica chimica acta}, volume = {1205}, number = {}, pages = {339541}, doi = {10.1016/j.aca.2022.339541}, pmid = {35414381}, issn = {1873-4324}, mesh = {*Biosensing Techniques ; CRISPR-Cas Systems ; Colorimetry ; Molecular Probes ; *Nucleic Acids ; }, abstract = {CRISPR/Cas-based analytical procedures have revolutionized the sensing platform to fulfill the requirements of the current era in terms of sensitivity, selectivity, robustness, user-friendly feature, and cost-effectiveness for the detection of nucleic acid as well as non-nucleic acid analytes. Molecular target monitoring and transduction of the signals is a crucial prerequisite for precise molecular sensing tools. Besides, the reporting systems have become the last milestone for fabricating Cas-based molecular probes to visualize the activation of CRISPR/Cas enzymes. In this review, we have highlighted various CRISPR/Cas reporters, their mechanisms, sensing strategies, merits, and demerits. Moreover, signal transducers, i.e. fluorescent, colorimetric, and electrochemical, have also been discussed in detail along with various sensing strategies to generate recordable signals. It was concluded that there is still a need to overcome issues offered by the reported sensing devices, such as off-target effect, target sequence limitation, multiplexed quantitative detection, the influence of the inhibitor, and reaction kinetic constraint. Additionally, it is required to make them available for commercial use by validating their stability, robustness, safety profile in an off-lab environment as most of the probes have been tested in the controlled atmosphere of the laboratories. We believe that this novel critical interpretation and summary will assist the researchers in designing and validating new CRISPR/Cas reporters and probes for practical applications on a commercial scale.}, }
@article {pmid35414240, year = {2022}, author = {Liu, Y and Champer, J}, title = {Modelling homing suppression gene drive in haplodiploid organisms.}, journal = {Proceedings. Biological sciences}, volume = {289}, number = {1972}, pages = {20220320}, pmid = {35414240}, issn = {1471-2954}, mesh = {Alleles ; CRISPR-Cas Systems ; Female ; *Gene Drive Technology/methods ; Germ Cells ; Humans ; Male ; RNA, Guide/genetics ; }, abstract = {Gene drives have shown great promise for suppression of pest populations. These engineered alleles can function by a variety of mechanisms, but the most common is the CRISPR homing drive, which converts wild-type alleles to drive alleles in the germline of heterozygotes. Some potential target species are haplodiploid, in which males develop from unfertilized eggs and thus have only one copy of each chromosome. This prevents drive conversion, a substantial disadvantage compared to diploids where drive conversion can take place in both sexes. Here, we study homing suppression gene drives in haplodiploids and find that a drive targeting a female fertility gene could still be successful. However, such drives are less powerful than in diploids and suffer more from functional resistance alleles. They are substantially more vulnerable to high resistance allele formation in the embryo owing to maternally deposited Cas9 and guide RNA and also to somatic cleavage activity. Examining spatial models where organisms move over a continuous landscape, we find that haplodiploid suppression drives surprisingly perform nearly as well as in diploids, possibly owing to their ability to spread further before inducing strong suppression. Together, these results indicate that gene drive can potentially be used to effectively suppress haplodiploid populations.}, }
@article {pmid35414130, year = {2022}, author = {Tornabene, P and Ferla, R and Llado-Santaeularia, M and Centrulo, M and Dell'Anno, M and Esposito, F and Marrocco, E and Pone, E and Minopoli, R and Iodice, C and Nusco, E and Rossi, S and Lyubenova, H and Manfredi, A and Di Filippo, L and Iuliano, A and Torella, A and Piluso, G and Musacchia, F and Surace, EM and Cacchiarelli, D and Nigro, V and Auricchio, A}, title = {Therapeutic homology-independent targeted integration in retina and liver.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {1963}, pmid = {35414130}, issn = {2041-1723}, support = {TA-GT-0619-0762-FED//Foundation Fighting Blindness (Foundation Fighting Blindness, Inc.)/ ; }, mesh = {Animals ; CRISPR-Cas Systems ; *Dependovirus/genetics ; *Gene Editing/methods ; Genetic Vectors/genetics ; Liver ; Mice ; Retina/metabolism ; Swine ; }, abstract = {Challenges to the widespread application of gene therapy with adeno-associated viral (AAV) vectors include dominant conditions due to gain-of-function mutations which require allele-specific knockout, as well as long-term transgene expression from proliferating tissues, which is hampered by AAV DNA episomal status. To overcome these challenges, we used CRISPR/Cas9-mediated homology-independent targeted integration (HITI) in retina and liver as paradigmatic target tissues. We show that AAV-HITI targets photoreceptors of both mouse and pig retina, and this results in significant improvements to retinal morphology and function in mice with autosomal dominant retinitis pigmentosa. In addition, we show that neonatal systemic AAV-HITI delivery achieves stable liver transgene expression and phenotypic improvement in a mouse model of a severe lysosomal storage disease. We also show that HITI applications predominantly result in on-target editing. These results lay the groundwork for the application of AAV-HITI for the treatment of diseases affecting various organs.}, }
@article {pmid35414049, year = {2022}, author = {Shi, H and Xu, Y and Tian, N and Yang, M and Liang, FS}, title = {Inducible and reversible RNA N6-methyladenosine editing.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {1958}, pmid = {35414049}, issn = {2041-1723}, support = {R21 CA247638/CA/NCI NIH HHS/United States ; R21CA247638//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; }, mesh = {Adenosine/analogs &amp; derivatives ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Epigenesis, Genetic ; Humans ; *RNA/genetics ; }, abstract = {RNA modifications, including N6-methyladenosine (m6A), have been reported to regulate fundamental RNA processes and properties, and directly linked to various human diseases. Methods enabling temporal and transcript/locus-specific editing of specific RNA modifications are essential, but still limited, to dissect the dynamic and context-dependent functions of these epigenetic modifications. Here, we develop a chemically inducible and reversible RNA m6A modification editing platform integrating chemically induced proximity (CIP) and CRISPR methods. We show that m6A editing can be temporally controlled at specific sites of individual RNA transcripts by the addition or removal of the CIP inducer, abscisic acid (ABA), in the system. By incorporating a photo-caged ABA, a light-controlled version of m6A editing platform can be developed. We expect that this platform and strategy can be generally applied to edit other RNA modifications in addition to m6A.}, }
@article {pmid35414015, year = {2022}, author = {Guo, LY and Bian, J and Davis, AE and Liu, P and Kempton, HR and Zhang, X and Chemparathy, A and Gu, B and Lin, X and Rane, DA and Xu, X and Jamiolkowski, RM and Hu, Y and Wang, S and Qi, LS}, title = {Multiplexed genome regulation in vivo with hyper-efficient Cas12a.}, journal = {Nature cell biology}, volume = {24}, number = {4}, pages = {590-600}, pmid = {35414015}, issn = {1476-4679}, support = {2046650//NSF | BIO | Division of Molecular and Cellular Biosciences (MCB)/ ; P30 EY026877/EY/NEI NIH HHS/United States ; T32 EY020485/EY/NEI NIH HHS/United States ; DISC2-12669//California Institute for Regenerative Medicine (CIRM)/ ; R01 EY028106/EY/NEI NIH HHS/United States ; R01 EY032518/EY/NEI NIH HHS/United States ; R01 EY023295/EY/NEI NIH HHS/United States ; P30-EY026877//U.S. Department of Health &amp; Human Services | NIH | NIH Office of the Director (OD)/ ; R01 NS109990/NS/NINDS NIH HHS/United States ; R01 EY024932/EY/NEI NIH HHS/United States ; U01 DK127405/DK/NIDDK NIH HHS/United States ; }, mesh = {Animals ; *CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Editing ; Mice ; RNA/metabolism ; }, abstract = {Multiplexed modulation of endogenous genes is crucial for sophisticated gene therapy and cell engineering. CRISPR-Cas12a systems enable versatile multiple-genomic-loci targeting by processing numerous CRISPR RNAs (crRNAs) from a single transcript; however, their low efficiency has hindered in vivo applications. Through structure-guided protein engineering, we developed a hyper-efficient Lachnospiraceae bacterium Cas12a variant, termed hyperCas12a, with its catalytically dead version hyperdCas12a showing significantly enhanced efficacy for gene activation, particularly at low concentrations of crRNA. We demonstrate that hyperdCas12a has comparable off-target effects compared with the wild-type system and exhibits enhanced activity for gene editing and repression. Delivery of the hyperdCas12a activator and a single crRNA array simultaneously activating the endogenous Oct4, Sox2 and Klf4 genes in the retina of post-natal mice alters the differentiation of retinal progenitor cells. The hyperCas12a system offers a versatile in vivo tool for a broad range of gene-modulation and gene-therapy applications.}, }
@article {pmid35413320, year = {2022}, author = {Chauhan, N and Saxena, K and Jain, U}, title = {Single molecule detection; from microscopy to sensors.}, journal = {International journal of biological macromolecules}, volume = {209}, number = {Pt A}, pages = {1389-1401}, doi = {10.1016/j.ijbiomac.2022.04.038}, pmid = {35413320}, issn = {1879-0003}, mesh = {*Biosensing Techniques/methods ; Microscopy ; *Nanopores ; Nanotechnology/methods ; }, abstract = {Single molecule detection is necessary to find out physical, chemical properties and their mechanism involved in the normal functioning of body cells. In this way, they can provide a new direction to the healthcare system. Various techniques have been developed and employed for their successful detection. Herein, we have emphasized various traditional methods as well as biosensing technology which offer single molecule sensitivity. The various methods including plasmonic resonance, nanopores, whispering gallery mode, Simoa assay and recognition tunneling are discussed in the initial part which has been followed by a discussion about biosensor-based detection. Plasmonic, SERS, CRISPR/Cas, and other types of biosensors are focused in this review and found to be highly sensitive for single molecule detection. This review provides an overview of progression in different techniques employed for single molecule detection.}, }
@article {pmid35412632, year = {2022}, author = {Pan, X and Li, H and Zhang, X}, title = {TedSim: temporal dynamics simulation of single-cell RNA sequencing data and cell division history.}, journal = {Nucleic acids research}, volume = {50}, number = {8}, pages = {4272-4288}, pmid = {35412632}, issn = {1362-4962}, support = {R35 GM143070/GM/NIGMS NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems ; Cell Division/genetics ; Cell Lineage/genetics ; Sequence Analysis, RNA/methods ; *Single-Cell Analysis/methods ; }, abstract = {Recently, lineage tracing technology using CRISPR/Cas9 genome editing has enabled simultaneous readouts of gene expressions and lineage barcodes, which allows for the reconstruction of the cell division tree and makes it possible to reconstruct ancestral cell types and trace the origin of each cell type. Meanwhile, trajectory inference methods are widely used to infer cell trajectories and pseudotime in a dynamic process using gene expression data of present-day cells. Here, we present TedSim (single-cell temporal dynamics simulator), which simulates the cell division events from the root cell to present-day cells, simultaneously generating two data modalities for each single cell: the lineage barcode and gene expression data. TedSim is a framework that connects the two problems: lineage tracing and trajectory inference. Using TedSim, we conducted analysis to show that (i) TedSim generates realistic gene expression and barcode data, as well as realistic relationships between these two data modalities; (ii) trajectory inference methods can recover the underlying cell state transition mechanism with balanced cell type compositions; and (iii) integrating gene expression and barcode data can provide more insights into the temporal dynamics in cell differentiation compared to using only one type of data, but better integration methods need to be developed.}, }
@article {pmid35412236, year = {2022}, author = {Aksenova, V and Arnaoutov, A and Dasso, M}, title = {Analysis of Nucleoporin Function Using Inducible Degron Techniques.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2502}, number = {}, pages = {129-150}, pmid = {35412236}, issn = {1940-6029}, mesh = {CRISPR-Cas Systems ; *Gene Targeting/methods ; *Indoleacetic Acids/pharmacology ; Nuclear Pore ; *Nuclear Pore Complex Proteins/chemistry/genetics/metabolism ; Proteins ; Proteolysis ; }, abstract = {Over the last decade, the use of auxin-inducible degrons (AID) to control the stability of target proteins has revolutionized the field of cell biology. AID-mediated degradation helps to overcome multiple hurdles that have been encountered in studying multisubunit protein complexes, like the nuclear pore complex (NPC), using classical biochemical and genetic methods. We have used the AID system for acute depletion of individual members of the NPC, called nucleoporins, in order to distinguish their roles both within established NPCs and during NPC assembly.Here, we describe a protocol for CRISPR/Cas9-mediated gene targeting of genes with the AID tag. As an example, we describe a step-by-step protocol for targeting of the NUP153 gene. We also provide recommendations for screening strategies and integration of the sequence encoding the Transport Inhibitor Response 1 (TIR1) protein, a E3-Ubiquitin ligase subunit necessary for AID-dependent protein degradation. In addition, we discuss applications of the NUP-AID system and functional assays for analysis of NUP-AID tagged cell lines.}, }
@article {pmid35411846, year = {2022}, author = {Rangarajan, AA and Yilmaz, C and Schnetz, K}, title = {Deletion of FRT-sites by no-SCAR recombineering in Escherichia coli.}, journal = {Microbiology (Reading, England)}, volume = {168}, number = {4}, pages = {}, doi = {10.1099/mic.0.001173}, pmid = {35411846}, issn = {1465-2080}, mesh = {*CRISPR-Cas Systems ; DNA, Single-Stranded ; *Escherichia coli/genetics ; Genetic Engineering/methods ; }, abstract = {Lambda-Red recombineering is the most commonly used method to create point mutations, insertions or deletions in Escherichia coli and other bacteria, but usually an Flp recognition target (FRT) scar-site is retained in the genome. Alternative scarless recombineering methods, including CRISPR/Cas9-assisted methods, generally require cloning steps and/or complex PCR schemes for specific targeting of the genome. Here we describe the deletion of FRT scar-sites by the scarless Cas9-assisted recombineering method no-SCAR using an FRT-specific guide RNA, sgRNAFRT, and locus-specific ssDNA oligonucleotides. We applied this method to construct a scarless E. coli strain suitable for gradual induction by l-arabinose. Genome sequencing of the resulting strain and its parent strains demonstrated that no additional mutations were introduced along with the simultaneous deletion of two FRT scar-sites. The FRT-specific no-SCAR selection by sgRNAFRT/Cas9 may be generally applicable to cure FRT scar-sites of E. coli strains constructed by classical λ-Red recombineering.}, }
@article {pmid35411005, year = {2022}, author = {Yang, L and Zhang, L and Yin, P and Ding, H and Xiao, Y and Zeng, J and Wang, W and Zhou, H and Wang, Q and Zhang, Y and Chen, Z and Yang, M and Feng, Y}, title = {Insights into the inhibition of type I-F CRISPR-Cas system by a multifunctional anti-CRISPR protein AcrIF24.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {1931}, pmid = {35411005}, issn = {2041-1723}, mesh = {*Bacteriophages/genetics/metabolism ; *CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems ; DNA/metabolism ; Viral Proteins/metabolism ; }, abstract = {CRISPR-Cas systems are prokaryotic adaptive immune systems and phages use anti-CRISPR proteins (Acrs) to counteract these systems. Here, we report the structures of AcrIF24 and its complex with the crRNA-guided surveillance (Csy) complex. The HTH motif of AcrIF24 can bind the Acr promoter region and repress its transcription, suggesting its role as an Aca gene in self-regulation. AcrIF24 forms a homodimer and further induces dimerization of the Csy complex. Apart from blocking the hybridization of target DNA to the crRNA, AcrIF24 also induces the binding of non-sequence-specific dsDNA to the Csy complex, similar to AcrIF9, although this binding seems to play a minor role in AcrIF24 inhibitory capacity. Further structural and biochemical studies of the Csy-AcrIF24-dsDNA complexes and of AcrIF24 mutants reveal that the HTH motif of AcrIF24 and the PAM recognition loop of the Csy complex are structural elements essential for this non-specific dsDNA binding. Moreover, AcrIF24 and AcrIF9 display distinct characteristics in inducing non-specific DNA binding. Together, our findings highlight a multifunctional Acr and suggest potential wide distribution of Acr-induced non-specific DNA binding.}, }
@article {pmid35410430, year = {2022}, author = {Che, P and Wu, E and Simon, MK and Anand, A and Lowe, K and Gao, H and Sigmund, AL and Yang, M and Albertsen, MC and Gordon-Kamm, W and Jones, TJ}, title = {Wuschel2 enables highly efficient CRISPR/Cas-targeted genome editing during rapid de novo shoot regeneration in sorghum.}, journal = {Communications biology}, volume = {5}, number = {1}, pages = {344}, pmid = {35410430}, issn = {2399-3642}, mesh = {CRISPR-Cas Systems ; Edible Grain/genetics ; *Gene Editing/methods ; Plants, Genetically Modified/genetics ; Regeneration/genetics ; *Sorghum/genetics ; }, abstract = {For many important crops including sorghum, use of CRISPR/Cas technology is limited not only by the delivery of the gene-modification components into a plant cell, but also by the ability to regenerate a fertile plant from the engineered cell through tissue culture. Here, we report that Wuschel2 (Wus2)-enabled transformation increases not only the transformation efficiency, but also the CRISPR/Cas-targeted genome editing frequency in sorghum (Sorghum bicolor L.). Using Agrobacterium-mediated transformation, we have demonstrated Wus2-induced direct somatic embryo formation and regeneration, bypassing genotype-dependent callus formation and significantly shortening the tissue culture cycle time. This method also increased the regeneration capacity that resulted in higher transformation efficiency across different sorghum varieties. Subsequently, advanced excision systems and "altruistic" transformation technology have been developed to generate high-quality morphogenic gene-free and/or selectable marker-free sorghum events. Finally, we demonstrate up to 6.8-fold increase in CRISPR/Cas9-mediated gene dropout frequency using Wus2-enabled transformation, compared to without Wus2, across various targeted loci in different sorghum genotypes.}, }
@article {pmid35410423, year = {2022}, author = {Liu, Y and Pinto, F and Wan, X and Yang, Z and Peng, S and Li, M and Cooper, JM and Xie, Z and French, CE and Wang, B}, title = {Reprogrammed tracrRNAs enable repurposing of RNAs as crRNAs and sequence-specific RNA biosensors.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {1937}, pmid = {35410423}, issn = {2041-1723}, support = {/WT_/Wellcome Trust/United Kingdom ; MR/S018875/1/MRC_/Medical Research Council/United Kingdom ; MR/V035401/1/MRC_/Medical Research Council/United Kingdom ; /DH_/Department of Health/United Kingdom ; }, mesh = {*Biosensing Techniques ; *COVID-19 ; CRISPR-Cas Systems/genetics ; Humans ; RNA, Guide/genetics/metabolism ; RNA, Viral/genetics ; SARS-CoV-2/genetics ; }, abstract = {In type II CRISPR systems, the guide RNA (gRNA) comprises a CRISPR RNA (crRNA) and a hybridized trans-acting CRISPR RNA (tracrRNA), both being essential in guided DNA targeting functions. Although tracrRNAs are diverse in sequence and structure across type II CRISPR systems, the programmability of crRNA-tracrRNA hybridization for Cas9 is not fully understood. Here, we reveal the programmability of crRNA-tracrRNA hybridization for Streptococcus pyogenes Cas9, and in doing so, redefine the capabilities of Cas9 proteins and the sources of crRNAs, providing new biosensing applications for type II CRISPR systems. By reprogramming the crRNA-tracrRNA hybridized sequence, we show that engineered crRNA-tracrRNA interactions can not only enable the design of orthogonal cellular computing devices but also facilitate the hijacking of endogenous small RNAs/mRNAs as crRNAs. We subsequently describe how these re-engineered gRNA pairings can be implemented as RNA sensors, capable of monitoring the transcriptional activity of various environment-responsive genomic genes, or detecting SARS-CoV-2 RNA in vitro, as an Atypical gRNA-activated Transcription Halting Alarm (AGATHA) biosensor.}, }
@article {pmid35410288, year = {2022}, author = {Oh, Y and Lee, WJ and Hur, JK and Song, WJ and Lee, Y and Kim, H and Gwon, LW and Kim, YH and Park, YH and Kim, CH and Lim, KS and Song, BS and Huh, JW and Kim, SU and Jun, BH and Jung, C and Lee, SH}, title = {Expansion of the prime editing modality with Cas9 from Francisella novicida.}, journal = {Genome biology}, volume = {23}, number = {1}, pages = {92}, pmid = {35410288}, issn = {1474-760X}, support = {NRF-2019R1C1C1006603//National Research Foundation/ ; NRF-2020R1I1A2075393//National Research Foundation/ ; NRF-2021M3A9I4024452//National Research Foundation/ ; 20009707//Ministry of Trade, Industry and Energy/ ; 9991006929, KMDF_PR_20200901_ 0264//Ministry of Trade, Industry and Energy/ ; HY-2020//the research fund of Hanyang University/ ; KGM1051911//Korea Research Institute of Bioscience and Biotechnology/ ; KGM4252122//Korea Research Institute of Bioscience and Biotechnology/ ; KGM5382113//Korea Research Institute of Bioscience and Biotechnology/ ; KGM4562121//Korea Research Institute of Bioscience and Biotechnology/ ; KGM5282113//Korea Research Institute of Bioscience and Biotechnology/ ; }, mesh = {*CRISPR-Cas Systems ; Deoxyribonuclease I/metabolism ; Francisella ; *Gene Editing ; Humans ; RNA-Directed DNA Polymerase ; }, abstract = {Prime editing can induce a desired base substitution, insertion, or deletion in a target gene using reverse transcriptase after nick formation by CRISPR nickase. In this study, we develop a technology that can be used to insert or replace external bases in the target DNA sequence by linking reverse transcriptase to the Francisella novicida Cas9, which is a CRISPR-Cas9 ortholog. Using FnCas9(H969A) nickase, the targeting limitation of existing Streptococcus pyogenes Cas9 nickase [SpCas9(H840A)]-based prime editing is dramatically extended, and accurate prime editing is induced specifically for the target genes in human cell lines.}, }
@article {pmid35409000, year = {2022}, author = {Huang, L and Luo, J and Song, N and Gao, W and Zhu, L and Yao, W}, title = {CRISPR/Cas9-Mediated Knockout of miR-130b Affects Mono- and Polyunsaturated Fatty Acid Content via PPARG-PGC1α Axis in Goat Mammary Epithelial Cells.}, journal = {International journal of molecular sciences}, volume = {23}, number = {7}, pages = {}, pmid = {35409000}, issn = {1422-0067}, support = {31772575//National Natural Science Foundation of China/ ; }, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Epithelial Cells/metabolism ; Fatty Acids/metabolism ; Fatty Acids, Unsaturated/metabolism ; *Goats/genetics/metabolism ; Mammary Glands, Animal/metabolism ; *MicroRNAs/genetics/metabolism ; PPAR gamma/metabolism ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/genetics/metabolism ; Sterol Regulatory Element Binding Protein 1/genetics/metabolism ; }, abstract = {MicroRNA (miRNA)-130b, as a regulator of lipid metabolism in adipose and mammary gland tissues, is actively involved in lipogenesis, but its endogenous role in fatty acid synthesis remains unclear. Here, we aimed to explore the function and underlying mechanism of miR-130b in fatty acid synthesis using the CRISPR/Cas9 system in primary goat mammary epithelial cells (GMEC). A single clone with deletion of 43 nucleotides showed a significant decrease in miR-130b-5p and miR-130b-3p abundances and an increase of target genes PGC1α and PPARG. In addition, knockout of miR-130b promoted triacylglycerol (TAG) and cholesterol accumulation, and decreased the proportion of monounsaturated fatty acids (MUFA) C16:1, C18:1 and polyunsaturated fatty acids (PUFA) C18:2, C20:3, C20:4, C20:5, C22:6. Similarly, the abundance of fatty acid synthesis genes ACACA and FASN and transcription regulators SREBP1c and SREBP2 was elevated. Subsequently, interference with PPARG instead of PGC1α in knockout cells restored the effect of miR-130b knockout, suggesting that PPARG is responsible for miR-130b regulating fatty acid synthesis. Moreover, disrupting PPARG inhibits PGC1α transcription and translation. These results reveal that miR-130b directly targets the PPARG-PGC1α axis, to inhibit fatty acid synthesis in GMEC. In conclusion, miR-130b could be a potential molecular regulator for improving the beneficial fatty acids content in goat milk.}, }
@article {pmid35408979, year = {2022}, author = {Tripathi, L and Ntui, VO and Tripathi, JN}, title = {Control of Bacterial Diseases of Banana Using CRISPR/Cas-Based Gene Editing.}, journal = {International journal of molecular sciences}, volume = {23}, number = {7}, pages = {}, pmid = {35408979}, issn = {1422-0067}, support = {CGIAR Window//United States Agency for International Development (USAID)/ ; CRP-RTB//CGIAR Research Program on Roots, Tubers and Bananas/ ; }, mesh = {*Bacterial Infections/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; *Musa/genetics/microbiology ; Plant Diseases/genetics/microbiology/prevention &amp; control ; *Xanthomonas/genetics ; }, abstract = {Banana is an important staple food crop and a source of income for smallholder farmers in about 150 tropical and sub-tropical countries. Several bacterial diseases, such as banana Xanthomonas wilt (BXW), blood, and moko disease, cause substantial impacts on banana production. There is a vast yield gap in the production of bananas in regions where bacterial pathogens and several other pathogens and pests are present together in the same field. BXW disease caused by Xanthomonas campestris pv. musacearum is reported to be the most destructive banana disease in East Africa. The disease affects all the banana varieties grown in the region. Only the wild-type diploid banana, Musa balbisiana, is resistant to BXW disease. Developing disease-resistant varieties of bananas is one of the most effective strategies to manage diseases. Recent advances in CRISPR/Cas-based gene editing techniques can accelerate banana improvement. Some progress has been made to create resistance against bacterial pathogens using CRISPR/Cas9-mediated gene editing by knocking out the disease-causing susceptibility (S) genes or activating the expression of the plant defense genes. A synopsis of recent advancements and perspectives on the application of gene editing for the control of bacterial wilt diseases are presented in this article.}, }
@article {pmid35403388, year = {2022}, author = {Pan, W and Cheng, Z and Han, Z and Yang, H and Zhang, W and Zhang, H}, title = {Efficient genetic transformation and CRISPR/Cas9-mediated genome editing of watermelon assisted by genes encoding developmental regulators.}, journal = {Journal of Zhejiang University. Science. B}, volume = {23}, number = {4}, pages = {339-344}, pmid = {35403388}, issn = {1862-1783}, support = {ZR202103010168//the Excellent Youth Foundation of Shandong Scientific Committee/ ; 2021T140017//the Shandong Science and Technology Innovation Funds, and the China Postdoctoral Science Foundation/ ; }, mesh = {CRISPR-Cas Systems ; *Citrullus/genetics ; *Cucurbitaceae/genetics ; Gene Editing ; Plant Breeding ; Transformation, Genetic ; }, abstract = {Cucurbitaceae is an important family of flowering plants containing multiple species of important food plants, such as melons, cucumbers, squashes, and pumpkins. However, a highly efficient genetic transformation system has not been established for most of these species (Nanasato and Tabei, 2020). Watermelon (Citrullus lanatus), an economically important and globally cultivated fruit crop, is a model species for fruit quality research due to its rich diversity of fruit size, shape, flavor, aroma, texture, peel and flesh color, and nutritional composition (Guo et al., 2019). Through pan-genome sequencing, many candidate loci associated with fruit quality traits have been identified (Guo et al., 2019). However, few of these loci have been validated. The major barrier is the low transformation efficiency of the species, with only few successful cases of genetic transformation reported so far (Tian et al., 2017; Feng et al., 2021; Wang JF et al., 2021; Wang YP et al., 2021). For example, Tian et al. (2017) obtained only 16 transgenic lines from about 960 cotyledon fragments, yielding a transformation efficiency of 1.67%. Therefore, efficient genetic transformation could not only facilitate the functional genomic studies in watermelon as well as other horticultural species, but also speed up the transgenic and genome-editing breeding.}, }
@article {pmid35400359, year = {2022}, author = {Wang, LY and Jiang, PF and Li, JZ and Hu, JD}, title = {[Effect of MiR-155 Knockout Mediated by Dual sgRNAs on Drug Sensitivity of FLT3-ITD+AML].}, journal = {Zhongguo shi yan xue ye xue za zhi}, volume = {30}, number = {2}, pages = {}, doi = {10.19746/j.cnki.issn.1009-2137.2022.02.002}, pmid = {35400359}, issn = {1009-2137}, mesh = {CRISPR-Cas Systems ; Doxorubicin/pharmacology ; Drug Resistance ; Gene Editing ; Humans ; *Leukemia, Myeloid, Acute/genetics ; *MicroRNAs/genetics ; RNA, Guide/genetics ; fms-Like Tyrosine Kinase 3/genetics ; }, abstract = {OBJECTIVE: Two sgRNAs transfected FLT3-ITD+AML cell line MV411 with different binding sites were introduced into CRISPR/cas9 to obtain MV411 cells with miR-155 gene knockout. To compare the efficiency of miR-155 gene knockout by single and double sgRNA transfection and their effects on cell phenotypes.<br><br>METHODS: The lentiviral vectors were generated containing either single sgRNA or dual sgRNAs and packaged into lentivirus particles. PCR was conducted to measure gene editing efficiency, and miR-155 expression was evaluated by qPCR. CCK-8 assay was used to evaluate the cell proliferation, and calculate drug sensitivity of cells to adriamycin and quizartinib. Annexin V-APC/7-AAD staining was used to label cell apoptosis induced by adriamycin and quizartinib.<br><br>RESULTS: In the dual sgRNAs transfected cells, a cleavage band could be observed, meaning the success of gene editing. Compared with the single sgRNA transfected MV411 cells, the expression level of mature miR-155-5p was lower in the dual sgRNA transfected cells. And, dual sgRNA transfected MV411 were more sensitive to adriamycin and quizartinib with lower IC50 and higher apoptosis rate.<br><br>CONCLUSION: The inhibition rate of miR-155 gene expression transfected by dual sgRNA is higher than that by single sgRNA. Dual sgRNA transfection can inhibit cell proliferation, reverse drug resistance, and induce apoptosis more significantly. Compared with single sgRNA transfection, dual sgRNA transfection is a highly efficient gene editing scheme.}, }
@article {pmid35398275, year = {2022}, author = {Kreuter, J and Stark, G and Mach, RL and Mach-Aigner, AR and Zimmermann, C}, title = {Fast and efficient CRISPR-mediated genome editing in Aureobasidium using Cas9 ribonucleoproteins.}, journal = {Journal of biotechnology}, volume = {350}, number = {}, pages = {11-16}, doi = {10.1016/j.jbiotec.2022.03.017}, pmid = {35398275}, issn = {1873-4863}, mesh = {*Ascomycota/genetics ; Aureobasidium ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Ribonucleoproteins/genetics ; Saccharomyces cerevisiae/genetics ; }, abstract = {Species of the genus Aureobasidium are ubiquitous, polyextremotolerant, "yeast-like" ascomycetes used for the industrial production of pullulan and other products and as biocontrol agents in agriculture. Their application potential and wide-spread occurrence make Aureobasidium spp. interesting study objects. The availability of a fast and efficient genome editing method is an obvious advantage for future basic and applied research on Aureobasidium. In this study, we describe the development of a CRISPR/Cas9-based genome editing method using ribonucleoproteins (RNPs) in A. pullulans and A. melanogenum. We demonstrate that this method can be used for single and multiplex genome editing using only RNPs by targeting URA3 (encoding for orotidine-5'-phosphate decarboxylase), ADE2 (encoding for phosphoribosylaminoimidazole carboxylase) and ARG4 (encoding for argininosuccinate lyase). We demonstrate the applicability of Trichoderma reesei pyr4 and Aspergillus fumigatus pyrG to complement the URA3 deficiency. Further, we show that using RNPs improves the homologous recombination rate and 20 bp long homologous flanks are sufficient. Therefore, the repair cassettes can be constructed by a single PCR, abolishing the need for laborious and time-consuming cloning, which is necessary for previously described methods for CRISPR-mediated genome editing in these fungi. The here presented method allows fast and efficient genome editing for gene deletions, modifications, and insertions in Auresobasidium with a minimized risk of off-target effects.}, }
@article {pmid35396804, year = {2022}, author = {Raghav, D and Jyoti, A and Siddiqui, AJ and Saxena, J}, title = {Plant-associated endophytic fungi as potential bio-factories for extracellular enzymes: Progress, Challenges and Strain improvement with precision approaches.}, journal = {Journal of applied microbiology}, volume = {}, number = {}, pages = {}, doi = {10.1111/jam.15574}, pmid = {35396804}, issn = {1365-2672}, abstract = {There is an intricate network of relations between endophytic fungi and their hosts that affects the production of various bioactive compounds. Plant-associated endophytic fungi contain industrially important enzymes and have the potential to fulfil their rapid demand in the international market to boost business in technology. Being safe and metabolically active, they have replaced the usage of toxic and harmful chemicals and hold a credible application in biotransformation, bioremediation and industrial processes. Despite these, there are limited reports on fungal endophytes that can directly cater to the demand and supply of industrially stable enzymes. The underlying reasons include low endogenous production and secretion of enzymes from fungal endophytes which have raised concern for widely accepted applications. Hence, it is imperative to augment the biosynthetic and secretory potential of fungal endophytes. Modern state-of-the-art biotechnological technologies aiming at strain improvement using cell factory engineering as well as precise gene editing like Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and its Associated proteins (Cas) systems which can provide a boost in fungal endophyte enzyme production. Additionally, it is vital to characterize optimum conditions to grow one strain with multiple enzymes (OSME). The present review encompasses various plants-derived endophytic fungal enzymes and their applications in various sectors. Furthermore, we postulate the feasibility of new precision approaches with an aim for strain improvement and enhanced enzyme production.}, }
@article {pmid35396760, year = {2022}, author = {Bhatt, A and Fatima, Z and Ruwali, M and Misra, CS and Rangu, SS and Rath, D and Rattan, A and Hameed, S}, title = {CLEVER assay: A visual and rapid RNA extraction-free detection of SARS-CoV-2 based on CRISPR-Cas integrated RT-LAMP technology.}, journal = {Journal of applied microbiology}, volume = {}, number = {}, pages = {}, doi = {10.1111/jam.15571}, pmid = {35396760}, issn = {1365-2672}, support = {BRNS/37080//Board of Research in Nuclear Sciences/ ; }, abstract = {AIM: The current scenario of COVID-19 pandemic has presented an almost insurmountable challenge even for the most sophisticated hospitals equipped with modern biomedical technology. There is an urgency to develop simple, fast and highly accurate methods for the rapid identification and isolation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected patients. To address the ongoing challenge, the present study offers a CLEVER assay (CRISPR-Cas integrated RT-LAMP Easy, Visual and Extraction-free RNA) which will allow RNA extraction-free method to visually diagnose COVID-19. RNA extraction is a major hurdle in preventing rapid and large-scale screening of samples particularly in low-resource regions because of the logistics and costs involved.<br><br>METHOD AND RESULT: Herein, the visual SARS-CoV-2 detection method consists of RNA extraction-free method directly utilizing the patient's nasopharyngeal and oropharyngeal samples for reverse transcription loop-mediated isothermal amplification (RT-LAMP). Additionally, the assay also utilizes the integration of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas12-based system using different guide RNAs of N, E and an internal control POP7 (human RNase P) genes along with visual detection via lateral flow readout-based dip sticks with unaided eye (~100 min). Overall, the clinical sensitivity and specificity of the CLEVER assay were 89.6% and 100%, respectively.<br><br>CONCLUSION: Together, our CLEVER assay offers a point-of-care tool with no equipment dependency and minimum technical expertise requirement for COVID-19 diagnosis.<br><br>To address the challenges associated with COVID-19 diagnosis, we need a faster, direct and more versatile detection method for an efficient epidemiological management of the COVID-19 outbreak. The present study involves developing a method for detection of SARS-CoV-2 in human body without RNA isolation step that can visually be detected with unaided eye. Taken together, our assay offers to overcome one major defect of the prior art, that is, RNA extraction step, which could limit the deployment of the previous assays in a testing site having limited lab infrastructure.}, }
@article {pmid35395997, year = {2022}, author = {Yang, F and Jiang, M and Lin, ZH and Xie, ZL and Ma, ZN and Yang, L and Liu, H and Wang, ZY and Zhou, L}, title = {[Effects of the ITGA2B Nonsense Mutation (c.2659C &gt; T, p.Q887X) on Platelet Function in a Mouse Model of Glanzmann's Thrombasthenia Generated with CRISPR/Cas9 Technology].}, journal = {Zhongguo shi yan xue ye xue za zhi}, volume = {30}, number = {2}, pages = {559-564}, doi = {10.19746/j.cnki.issn.1009-2137.2022.02.040}, pmid = {35395997}, issn = {1009-2137}, mesh = {Animals ; CRISPR-Cas Systems ; *Codon, Nonsense ; Disease Models, Animal ; Fibrinogen/genetics ; Humans ; *Integrin alpha2/genetics ; Mice ; Oligonucleotides ; Platelet Glycoprotein GPIIb-IIIa Complex/genetics ; RNA, Guide ; *Thrombasthenia/diagnosis/genetics ; Thrombin/genetics ; }, abstract = {OBJECTIVE: To construct a mouse model of Glanzmann's thrombasthenia (GT) with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation by CRISPR/Cas9 technology, and then further explore the expression and function of glycoprotein αIIbβ3 on the surface of platelet membrane.<br><br>METHODS: The donor oligonucleotide and gRNA vector were designed and synthesized according to the ITGA2B gene sequence. The gRNA and Cas9 mRNA were injected into fertilized eggs with donor oligonucleotide and then sent back to the oviduct of surrogate mouse. Positive F0 mice were confirmed by PCR genotyping and sequence analysis after birth. The F1 generation of heterozygous GT mice were obtained by PCR and sequencing from F0 bred with WT mice, and then homozygous GT mice and WT mice were obtained by mating with each other. The phenotype of the model was then further verified by detecting tail hemorrhage time, saphenous vein bleeding time, platelet aggregation, expression and function of αIIbβ3 on the surface of platelet.<br><br>RESULTS: The bleeding time of GT mice was significantly longer than that of WT mice (P<0.01). Induced by collagen, thrombin, and adenosine diphosphate (ADP), platelet aggregation in GT mice was significantly inhibited (P<0.01, P<0.01, P<0.05). Flow cytometry analysis showed that the expression of αIIbβ3 on the platelet surface of GT mice decreased significantly compared with WT mice (P<0.01), and binding amounts of activated platelets to fibrinogen were significantly reduced after thrombin stimulation (P<0.01). The spreading area of platelet on fibrinogen in GT mice was significantly smaller than that in WT mice (P<0.05).<br><br>CONCLUSION: A GT mouse model with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation has been established successfully by CRISPR/Cas9 technology. The aggregation function of platelet in this model is defective, which is consistent with GT performance.}, }
@article {pmid35395959, year = {2022}, author = {Wang, LY and Jiang, PF and Li, JZ and Hu, JD}, title = {[Effect of MiR-155 Knockout Mediated by Dual sgRNAs on Drug Sensitivity of FLT3-ITD+AML].}, journal = {Zhongguo shi yan xue ye xue za zhi}, volume = {30}, number = {2}, pages = {334-340}, doi = {10.19746/j.cnki.issn.1009-2137.2022.02.002}, pmid = {35395959}, issn = {1009-2137}, mesh = {CRISPR-Cas Systems ; Doxorubicin/pharmacology ; Drug Resistance ; Gene Editing ; Humans ; *Leukemia, Myeloid, Acute/genetics ; *MicroRNAs/genetics ; RNA, Guide/genetics ; fms-Like Tyrosine Kinase 3/genetics ; }, abstract = {OBJECTIVE: Two sgRNAs transfected FLT3-ITD+AML cell line MV411 with different binding sites were introduced into CRISPR/cas9 to obtain MV411 cells with miR-155 gene knockout. To compare the efficiency of miR-155 gene knockout by single and double sgRNA transfection and their effects on cell phenotypes.<br><br>METHODS: The lentiviral vectors were generated containing either single sgRNA or dual sgRNAs and packaged into lentivirus particles. PCR was conducted to measure gene editing efficiency, and miR-155 expression was evaluated by qPCR. CCK-8 assay was used to evaluate the cell proliferation, and calculate drug sensitivity of cells to adriamycin and quizartinib. Annexin V-APC/7-AAD staining was used to label cell apoptosis induced by adriamycin and quizartinib.<br><br>RESULTS: In the dual sgRNAs transfected cells, a cleavage band could be observed, meaning the success of gene editing. Compared with the single sgRNA transfected MV411 cells, the expression level of mature miR-155-5p was lower in the dual sgRNA transfected cells. And, dual sgRNA transfected MV411 were more sensitive to adriamycin and quizartinib with lower IC50 and higher apoptosis rate.<br><br>CONCLUSION: The inhibition rate of miR-155 gene expression transfected by dual sgRNA is higher than that by single sgRNA. Dual sgRNA transfection can inhibit cell proliferation, reverse drug resistance, and induce apoptosis more significantly. Compared with single sgRNA transfection, dual sgRNA transfection is a highly efficient gene editing scheme.}, }
@article {pmid35394860, year = {2022}, author = {Mamontov, V and Martynov, A and Morozova, N and Bukatin, A and Staroverov, DB and Lukyanov, KA and Ispolatov, Y and Semenova, E and Severinov, K}, title = {Persistence of plasmids targeted by CRISPR interference in bacterial populations.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {119}, number = {15}, pages = {e2114905119}, doi = {10.1073/pnas.2114905119}, pmid = {35394860}, issn = {1091-6490}, mesh = {*CRISPR-Cas Systems/genetics/physiology ; *Escherichia coli/genetics ; Gene-Environment Interaction ; *Interspersed Repetitive Sequences/genetics ; Models, Genetic ; *Plasmids/genetics ; }, abstract = {SignificanceRNA-guided CRISPR-Cas nucleases efficiently protect bacterial cells from phage infection and plasmid transformation. Yet, the efficiency of CRISPR-Cas defense is not absolute. Mutations in either CRISPR-Cas components of the host or mobile genetic elements regions targeted by CRISPR-Cas inactivate the defensive action. Here, we show that even at conditions of active CRISPR-Cas and unaltered targeted plasmids, a kinetic equilibrium between CRISPR-Cas nucleases action and plasmid replication processes allows for existence of a small subpopulation of plasmid-bearing cells on the background of cells that have been cured from the plasmid. In nature, the observed diversification of phenotypes may allow rapid changes in the population structure to meet the demands of the environment.}, }
@article {pmid35390717, year = {2022}, author = {Ivanov, AV and Safenkova, IV and Zherdev, AV and Dzantiev, BB}, title = {DIRECT2: A novel platform for a CRISPR-Cas12-based assay comprising universal DNA-IgG probe and a direct lateral flow test.}, journal = {Biosensors &amp; bioelectronics}, volume = {208}, number = {}, pages = {114227}, doi = {10.1016/j.bios.2022.114227}, pmid = {35390717}, issn = {1873-4235}, mesh = {Animals ; *Biosensing Techniques ; CRISPR-Cas Systems/genetics ; DNA/genetics ; DNA Probes/genetics ; DNA, Single-Stranded ; Gold ; Immunoglobulin G ; *Metal Nanoparticles ; Mice ; }, abstract = {CRISPR-Cas12-based biosensors are a promising tool for the detection of nucleic acids. After dsDNA-target-activated Cas12 cleaves the ssDNA probe, a lateral flow test (LFT) is applied for rapid, simple, and out-of-laboratory detection of the cleaved probe. However, most of the existing approaches of LFT detection have disadvantages related to inverted test/control zones in which the assay result depends not only on the cleavage of the probe but also on the second factor: the binding of the non-cleaved probe in the control zone. We proposed a novel platform for the detection of trans-cleaved DNA using a universal DNA-IgG probe and LFT with the sequential direct location of test and control zones. The advantage of the platform consists of the assay result depending only on the cleaved probe. For this, we designed a composite probe that comprise two parts: the DNA part (biotinylated dsDNA connected to ssDNA with fluorescein) (FAM), and the antibody part (mouse anti-FAM IgG). The Cas12, with guide RNA, was activated by the dsDNA-target. The activated Cas12 cleaved the probe, releasing the ssDNA-FAM-IgG reporter that was detected by the LFT. The sandwich LFT was proposed with anti-mouse IgG adsorbed in the test zone and on the surface of gold nanoparticles. We called the platform with direct location zones and direct analyte-signal dependence the DNA-Immunoglobulin Reporter Endonuclease Cleavage Test (DIRECT2). Therefore, this proof-of-concept study demonstrated that the combination of the proposed DNA-IgG probe and direct LFT opens new opportunities for CRISPR-Cas12 activity detection and its bioanalytical applications.}, }
@article {pmid35389616, year = {2022}, author = {Abdullah,  and Wang, P and Han, T and Liu, W and Ren, W and Wu, Y and Xiao, Y}, title = {Adenine Base Editing System for Pseudomonas and Prediction Workflow for Protein Dysfunction via ABE.}, journal = {ACS synthetic biology}, volume = {11}, number = {4}, pages = {1650-1657}, doi = {10.1021/acssynbio.2c00066}, pmid = {35389616}, issn = {2161-5063}, mesh = {Adenine/metabolism ; *CRISPR-Cas Systems ; *Gene Editing ; Pseudomonas/genetics/metabolism ; Workflow ; }, abstract = {Pseudomonas is a large genus that inhabits diverse environments due to its distinct metabolic versatility. Its applications range from environmental to industrial biotechnology. Molecular tools that allow precise and efficient genetic manipulation are required to understand and harness its full potential. Here, we report the development of a highly efficient adenine base editing system, i.e., dxABE-PS, for Pseudomonas species. The system allows A:T → G:C transition with up to 100% efficiency along a broad target spectrum because we use xCas9 3.7, which recognizes NG PAM. To enhance the dxABE-PS utility, we develop a prediction workflow for protein dysfunction using ABE, namely, DABE-CSP (dysfunction via ABE through CRISPOR-SIFT prediction). We applied DABE-CSP to inactivate several genes in Pseudomonas putida KT2440 to accumulate a nylon precursor, i.e., muconic acid from catechol with 100% yield. Moreover, we expanded the ABE to non-model Pseudomonas species by developing an nxABE system for P. chengduensisDY56-96, isolated from sediment samples from the seamount area in the West Pacific Ocean. Taken together, the establishment of the ABE systems along with DABE-CSP will fast-track research on Pseudomonas species.}, }
@article {pmid35389262, year = {2022}, author = {Martinez, MG and Combe, E and Inchauspe, A and Mangeot, PE and Delberghe, E and Chapus, F and Neveu, G and Alam, A and Carter, K and Testoni, B and Zoulim, F}, title = {CRISPR-Cas9 Targeting of Hepatitis B Virus Covalently Closed Circular DNA Generates Transcriptionally Active Episomal Variants.}, journal = {mBio}, volume = {13}, number = {2}, pages = {e0288821}, pmid = {35389262}, issn = {2150-7511}, support = {ANR-10-LABX-61//Labex DevWeCan/ ; }, mesh = {CRISPR-Cas Systems ; DNA, Circular/genetics ; DNA, Viral/genetics ; *Hepatitis B virus/genetics ; *Hepatitis B, Chronic/drug therapy ; Humans ; RNA, Guide/genetics ; }, abstract = {Chronic hepatitis B virus (HBV) infection persists due to the lack of therapies that effectively target the HBV covalently closed circular DNA (cccDNA). We used HBV-specific guide RNAs (gRNAs) and CRISPR-Cas9 and determined the fate of cccDNA after gene editing. We set up a ribonucleoprotein (RNP) delivery system in HBV-infected HepG2-NTCP cells. HBV parameters after Cas9 editing were analyzed. Southern blot (SB) analysis and DNA/RNA sequencing (DNA/RNA-seq) were performed to determine the consequences of cccDNA editing and transcriptional activity of mutated cccDNA. Treatment of infected cells with HBV-specific gRNAs showed that CRISPR-Cas9 can efficiently affect HBV replication. The appearance of episomal HBV DNA variants after dual gRNA treatment was observed by PCR, SB analysis, and DNA/RNA-seq. These transcriptionally active variants are the products of simultaneous Cas9-induced double-strand breaks in two target sites, followed by repair and religation of both short and long fragments. Following suppression of HBV DNA replicative intermediates by nucleoside analogs, mutations and formation of smaller transcriptionally active HBV variants were still observed, suggesting that established cccDNA is accessible to CRISPR-Cas9 editing. Targeting HBV DNA with CRISPR-Cas9 leads to cleavage followed by appearance of episomal HBV DNA variants. Effects induced by Cas9 were sustainable after RNP degradation/loss of detection, suggesting permanent changes in the HBV genome instead of transient effects due to transcriptional interference. IMPORTANCE Hepatitis B virus infection can develop into chronic infection, cirrhosis, and hepatocellular carcinoma. Treatment of chronic hepatitis B requires novel approaches to directly target the viral minichromosome, which is responsible for the persistence of the disease. Designer nuclease approaches represent a promising strategy to treat chronic infectious diseases; however, comprehensive knowledge about the fate of the HBV minichromosome is needed before this potent tool can be used as a potential therapeutic approach. This study provides an in-depth analysis of CRISPR-Cas9 targeting of HBV minichromosome.}, }
@article {pmid35389256, year = {2022}, author = {Govindarajan, S and Borges, A and Karambelkar, S and Bondy-Denomy, J}, title = {Distinct Subcellular Localization of a Type I CRISPR Complex and the Cas3 Nuclease in Bacteria.}, journal = {Journal of bacteriology}, volume = {204}, number = {5}, pages = {e0010522}, doi = {10.1128/jb.00105-22}, pmid = {35389256}, issn = {1098-5530}, support = {DP5-OD021344//NIH Director's Early Independence award/ ; //Sandler Foundation/ ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems are prokaryotic adaptive immune systems that have been well characterized biochemically, but in vivo spatiotemporal regulation and cell biology remain largely unaddressed. Here, we used fluorescent fusion proteins introduced at the chromosomal CRISPR-Cas locus to study the localization of the type I-F CRISPR-Cas system in Pseudomonas aeruginosa. When lacking a target in the cell, the Cascade complex is broadly nucleoid bound, while Cas3 is diffuse in the cytoplasm. When targeted to an integrated prophage, however, the CRISPR RNA (crRNA)-guided type I-F Cascade complex and a majority of Cas3 molecules in the cell are recruited to a single focus. Nucleoid association of the Csy proteins that form the Cascade complex is crRNA dependent and specifically inhibited by the expression of anti-CRISPR AcrIF2, which blocks protospacer adjacent motif (PAM) binding. The Cas9 nuclease is also nucleoid localized, only when single guide RNA (sgRNA) bound, which is abolished by the PAM-binding inhibitor AcrIIA4. Our findings reveal PAM-dependent nucleoid surveillance and spatiotemporal regulation in type I CRISPR-Cas that separates the nuclease-helicase Cas3 from the crRNA-guided surveillance complex. IMPORTANCE CRISPR-Cas systems, the prokaryotic adaptive immune systems, are largely understood using structural biology, biochemistry, and genetics. How CRISPR-Cas effectors are organized within cells is currently not well understood. By investigating the cell biology of the type I-F CRISPR-Cas system, we show that the surveillance complex, which "patrols" the cell to find targets, is largely nucleoid bound, while Cas3 nuclease is cytoplasmic. Nucleoid localization is also conserved for class 2 CRISPR-Cas single protein effector Cas9. Our observation of differential localization of the surveillance complex and Cas3 reveals a new layer of posttranslational spatiotemporal regulation to prevent autoimmunity.}, }
@article {pmid35389053, year = {2022}, author = {Töpfer, R and Trapp, O}, title = {A cool climate perspective on grapevine breeding: climate change and sustainability are driving forces for changing varieties in a traditional market.}, journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik}, volume = {}, number = {}, pages = {}, pmid = {35389053}, issn = {1432-2242}, abstract = {A multitude of diverse breeding goals need to be combined in a new cultivar, which always forces to compromise. The biggest challenge grapevine breeders face is the extraordinarily complex trait of wine quality, which is the all-pervasive and most debated characteristic. Since the 1920s, Germany runs continuous grapevine breeding programmes. This continuity was the key to success and lead to various new cultivars on the market, so called PIWIs. Initially, introduced pests and diseases such as phylloxera, powdery and downy mildew were the driving forces for breeding. However, preconceptions about the wine quality of new resistant selections impeded the market introduction. These preconceptions are still echoing today and may be the reason in large parts of the viticultural community for: (1) ignoring substantial breeding progress, and (2) sticking to successful markets of well-known varietal wines or blends (e.g. Chardonnay, Cabernet Sauvignon, Riesling). New is the need to improve viticulture´s sustainability and to adapt to changing environmental conditions. Climate change with its extreme weather will impose the need for a change in cultivars in many wine growing regions. Therefore, a paradigm shift is knocking on the door: new varieties (PIWIs) versus traditional varieties for climate adapted and sustainable viticulture. However, it will be slow process and viticulture is politically well advised to pave the way to variety innovation. In contrast to the widely available PIWIs, competitive cultivars created by means of new breeding technologies (NBT, e.g. through CRISPR/Cas) are still decades from introduction to the market.}, }
@article {pmid35388178, year = {2022}, author = {Rönspies, M and Schindele, P and Wetzel, R and Puchta, H}, title = {CRISPR-Cas9-mediated chromosome engineering in Arabidopsis thaliana.}, journal = {Nature protocols}, volume = {17}, number = {5}, pages = {1332-1358}, pmid = {35388178}, issn = {1750-2799}, mesh = {*Arabidopsis/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Chromosomes ; Gene Editing/methods ; Mutation ; RNA, Guide/genetics/metabolism ; }, abstract = {The rise of the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system has made it possible to induce double-strand breaks at almost any desired target site in the genome. In plant somatic cells, double-strand breaks are predominantly repaired by the error-prone nonhomologous end-joining pathway, which can lead to mutations at the break site upon repair. So far, it had only been possible to induce genomic changes of up to a few hundred kilobases in plants utilizing this mechanism. However, by combining the highly efficient Staphylococcus aureus Cas9 (SaCas9) with an egg-cell-specific promoter to facilitate heritable mutations, chromosomal rearrangements in the Mb range, such as inversion and translocations, were obtained in Arabidopsis thaliana recently. Here we describe the chromosome-engineering protocol used to generate these heritable chromosomal rearrangements in A. thaliana. The protocol is based on Agrobacterium-mediated transformation of A. thaliana with transfer DNA constructs containing SaCas9, which is driven by an egg-cell-specific promoter, and two guide RNAs that have been preselected based on their cutting efficiency. In the T1 generation, primary transformants are selected and, if required, analyzed by Droplet Digital PCR and propagated. In the following generations, junction-specific PCR screenings are carried out until plants that carry the rearrangement homozygously are identified. Using this protocol, overall rearrangement frequencies range between 0.03% and 0.5%, depending on the type of rearrangement. In total, it takes about 1 year to establish homozygous lines.}, }
@article {pmid35388146, year = {2022}, author = {Fuchs, RT and Curcuru, JL and Mabuchi, M and Noireterre, A and Weigele, PR and Sun, Z and Robb, GB}, title = {Characterization of Cme and Yme thermostable Cas12a orthologs.}, journal = {Communications biology}, volume = {5}, number = {1}, pages = {325}, pmid = {35388146}, issn = {2399-3642}, mesh = {*CRISPR-Cas Systems ; DNA/genetics ; *DNA Cleavage ; Nucleic Acid Conformation ; RNA, Guide/genetics/metabolism ; }, abstract = {CRISPR-Cas12a proteins are RNA-guided endonucleases that cleave invading DNA containing target sequences adjacent to protospacer adjacent motifs (PAM). Cas12a orthologs have been repurposed for genome editing in non-native organisms by reprogramming them with guide RNAs to target specific sites in genomic DNA. After single-turnover dsDNA target cleavage, multiple-turnover, non-specific single-stranded DNA cleavage in trans is activated. This property has been utilized to develop in vitro assays to detect the presence of specific DNA target sequences. Most applications of Cas12a use one of three well-studied enzymes. Here, we characterize the in vitro activity of two previously unknown Cas12a orthologs. These enzymes are active at higher temperatures than widely used orthologs and have subtle differences in PAM preference, on-target cleavage, and trans nuclease activity. Together, our results enable refinement of Cas12a-based in vitro assays especially when elevated temperature is desirable.}, }
@article {pmid35387989, year = {2022}, author = {Clow, PA and Du, M and Jillette, N and Taghbalout, A and Zhu, JJ and Cheng, AW}, title = {CRISPR-mediated multiplexed live cell imaging of nonrepetitive genomic loci with one guide RNA per locus.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {1871}, pmid = {35387989}, issn = {2041-1723}, support = {P30 CA034196/CA/NCI NIH HHS/United States ; R01 HG009900/HG/NHGRI NIH HHS/United States ; }, mesh = {CRISPR-Cas Systems/genetics ; Chromatin/genetics ; Chromosomes ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genomics ; *RNA, Guide/genetics ; }, abstract = {Three-dimensional (3D) structures of the genome are dynamic, heterogeneous and functionally important. Live cell imaging has become the leading method for chromatin dynamics tracking. However, existing CRISPR- and TALE-based genomic labeling techniques have been hampered by laborious protocols and are ineffective in labeling non-repetitive sequences. Here, we report a versatile CRISPR/Casilio-based imaging method that allows for a nonrepetitive genomic locus to be labeled using one guide RNA. We construct Casilio dual-color probes to visualize the dynamic interactions of DNA elements in single live cells in the presence or absence of the cohesin subunit RAD21. Using a three-color palette, we track the dynamic 3D locations of multiple reference points along a chromatin loop. Casilio imaging reveals intercellular heterogeneity and interallelic asynchrony in chromatin interaction dynamics, underscoring the importance of studying genome structures in 4D.}, }
@article {pmid35387980, year = {2022}, author = {Zhang, G and Liu, Y and Huang, S and Qu, S and Cheng, D and Yao, Y and Ji, Q and Wang, X and Huang, X and Liu, J}, title = {Enhancement of prime editing via xrRNA motif-joined pegRNA.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {1856}, pmid = {35387980}, issn = {2041-1723}, mesh = {*CRISPR-Cas Systems/genetics ; Cell Line ; *Gene Editing ; Genome ; }, abstract = {The prime editors (PEs) have shown great promise for precise genome modification. However, their suboptimal efficiencies present a significant technical challenge. Here, by appending a viral exoribonuclease-resistant RNA motif (xrRNA) to the 3'-extended portion of pegRNAs for their increased resistance against degradation, we develop an upgraded PE platform (xrPE) with substantially enhanced editing efficiencies in multiple cell lines. A pan-target average enhancement of up to 3.1-, 4.5- and 2.5-fold in given cell types is observed for base conversions, small deletions, and small insertions, respectively. Additionally, xrPE exhibits comparable edit:indel ratios and similarly minimal off-target editing as the canonical PE3. Of note, parallel comparison of xrPE to the most recently developed epegRNA-based PE system shows their largely equivalent editing performances. Our study establishes a highly adaptable platform of improved PE that shall have broad implications.}, }
@article {pmid35387453, year = {2022}, author = {Liu, FX and Cui, JQ and Park, H and Chan, KW and Leung, T and Tang, BZ and Yao, S}, title = {Isothermal Background-Free Nucleic Acid Quantification by a One-Pot Cas13a Assay Using Droplet Microfluidics.}, journal = {Analytical chemistry}, volume = {94}, number = {15}, pages = {5883-5892}, doi = {10.1021/acs.analchem.2c00067}, pmid = {35387453}, issn = {1520-6882}, mesh = {Biological Assay ; CRISPR-Cas Systems ; *Microfluidics ; Nucleic Acid Amplification Techniques/methods ; *Nucleic Acids ; Recombinases/metabolism ; }, abstract = {High sensitivity and specificity nucleic acid detection has been achieved by the Cas13a collateral effect in combination with a separate recombinase polymerase amplification (RPA). However, these emerging methods cannot provide accurate quantification of nucleic acids because the two-step assay performance may be compromised if the RPA and Cas13a reactions are simply unified in a single step. In this work, we first addressed the challenges associated with enzymatic incompatibility and the macromolecular crowding effect in the one-pot assay development, making the consolidated RPA-Cas13a assay a facile and robust diagnostic tool. Next, we found that the one-pot reaction cannot precisely quantify the targets at low concentrations. Thus, by leveraging droplet microfluidics, we converted the one-pot assay to a digital quantification format, termed Microfluidics-Enabled Digital Isothermal Cas13a Assay (MEDICA). Due to the droplet compartmentation, MEDICA greatly accelerates the reaction and enables relative detection in 10 min and the end-point quantification in 25 min. Moreover, MEDICA facilitates the droplet binarization for counting because of background-free signals generated by trans-cleavage reporting of Cas13a. Our clinical validation highlights that CRISPR-based isothermal assays are promising for the next generation of nucleic acid quantification methods.}, }
@article {pmid35386853, year = {2022}, author = {He, X and Zeng, XX}, title = {Immunotherapy and CRISPR Cas Systems: Potential Cure of COVID-19?.}, journal = {Drug design, development and therapy}, volume = {16}, number = {}, pages = {951-972}, pmid = {35386853}, issn = {1177-8881}, mesh = {Antibodies, Neutralizing ; *COVID-19/drug therapy/therapy ; *CRISPR-Cas Systems ; Disease Progression ; Humans ; Immunization, Passive ; Immunologic Factors ; SARS-CoV-2 ; }, abstract = {The COVID-19 has plunged the world into a pandemic that affected millions. The continually emerging new variants of concern raise the question as to whether the existing vaccines will continue to provide sufficient protection for individuals from SARS-CoV-2 during natural infection. This narrative review aims to briefly outline various immunotherapeutic options and discuss the potential of clustered regularly interspaced short palindromic repeat (CRISPR Cas system technology against COVID-19 treatment as specific cure. As the development of vaccine, convalescent plasma, neutralizing antibodies are based on the understanding of human immune responses against SARS-CoV-2, boosting human body immune responses in case of SARS-CoV-2 infection, immunotherapeutics seem feasible as specific cure against COVID-19 if the present challenges are overcome. In cell based therapeutics, apart from the high costs, risks and side effects, there are technical problems such as the production of sufficient potent immune cells and antibodies under limited time to treat the COVID-19 patients in mild conditions prior to progression into a more severe case. The CRISPR Cas technology could be utilized to refine the specificity and safety of CAR-T cells, CAR-NK cells and neutralizing antibodies against SARS-CoV-2 during various stages of the COVID-19 disease progression in infected individuals. Moreover, CRISPR Cas technology are proposed in hypotheses to degrade the viral RNA in order to terminate the infection caused by SARS-CoV-2. Thus personalized cocktails of immunotherapeutics and CRISPR Cas systems against COVID-19 as a strategy might prevent further disease progression and circumvent immunity escape.}, }
@article {pmid35386818, year = {2022}, author = {Jiang, Y and Hoenisch, RC and Chang, Y and Bao, X and Cameron, CE and Lian, XL}, title = {Robust genome and RNA editing via CRISPR nucleases in PiggyBac systems.}, journal = {Bioactive materials}, volume = {14}, number = {}, pages = {313-320}, pmid = {35386818}, issn = {2452-199X}, abstract = {CRISPR/Cas-mediated genome editing in human pluripotent stem cells (hPSCs) offers unprecedented opportunities for developing in vitro disease modeling, drug screening and cell-based therapies. To efficiently deliver the CRISPR components, here we developed two all-in-one vectors containing Cas9/gRNA and inducible Cas13d/gRNA cassettes for robust genome editing and RNA interference respectively. These vectors utilized the PiggyBac transposon system, which allows stable expression of CRISPR components in hPSCs. The Cas9 vector PB-CRISPR exhibited high efficiency (up to 99%) of inducing gene knockout in both protein-coding genes and long non-coding RNAs. The other inducible Cas13d vector achieved extremely high efficiency in RNA knockdown (98% knockdown for CD90) with optimized gRNA designs. Taken together, our PiggyBac CRISPR vectors can serve as powerful toolkits for studying gene functions in hPSCs.}, }
@article {pmid35386712, year = {2022}, author = {Scheller, SH and Rashad, Y and Saleh, FM and Willingham, KA and Reilich, A and Lin, D and Izadpanah, R and Alt, EU and Braun, SE}, title = {Biallelic, Selectable, Knock-in Targeting of CCR5 via CRISPR-Cas9 Mediated Homology Directed Repair Inhibits HIV-1 Replication.}, journal = {Frontiers in immunology}, volume = {13}, number = {}, pages = {821190}, pmid = {35386712}, issn = {1664-3224}, mesh = {Alleles ; CRISPR-Cas Systems ; *HIV Infections/genetics ; *HIV Seropositivity/genetics ; *HIV-1/genetics ; Humans ; Receptors, CCR5/genetics ; Virus Replication ; }, abstract = {Transplanting HIV-1 positive patients with hematopoietic stem cells homozygous for a 32 bp deletion in the chemokine receptor type 5 (CCR5) gene resulted in a loss of detectable HIV-1, suggesting genetically disrupting CCR5 is a promising approach for HIV-1 cure. Targeting the CCR5-locus with CRISPR-Cas9 was shown to decrease the amount of CCR5 expression and HIV-1 susceptibility in vitro as well as in vivo. Still, only the individuals homozygous for the CCR5-Δ32 frameshift mutation confer complete resistance to HIV-1 infection. In this study we introduce a mechanism to target CCR5 and efficiently select for cells with biallelic frameshift insertion, using CRISPR-Cas9 mediated homology directed repair (HDR). We hypothesized that cells harboring two different selectable markers (double positive), each in one allele of the CCR5 locus, would carry a frameshift mutation in both alleles, lack CCR5 expression and resist HIV-1 infection. Inducing double-stranded breaks (DSB) via CRISPR-Cas9 leads to HDR and integration of a donor plasmid. Double-positive cells were selected via fluorescence-activated cell sorting (FACS), and CCR5 was analyzed genetically, phenotypically, and functionally. Targeted and selected populations showed a very high frequency of mutations and a drastic reduction in CCR5 surface expression. Most importantly, double-positive cells displayed potent inhibition to HIV-1 infection. Taken together, we show that targeting cells via CRISPR-Cas9 mediated HDR enables efficient selection of mutant cells that are deficient for CCR5 and highly resistant to HIV-1 infection.}, }
@article {pmid35386307, year = {2022}, author = {Haider, MZ and Shabbir, MAB and Yaqub, T and Sattar, A and Maan, MK and Mahmood, S and Mehmood, T and Aslam, HB}, title = {CRISPR-Cas System: An Adaptive Immune System's Association with Antibiotic Resistance in Salmonella enterica Serovar Enteritidis.}, journal = {BioMed research international}, volume = {2022}, number = {}, pages = {9080396}, pmid = {35386307}, issn = {2314-6141}, mesh = {Ampicillin ; Animals ; Anti-Bacterial Agents/pharmacology ; CRISPR-Cas Systems/genetics ; Drug Resistance, Multiple, Bacterial ; Microbial Sensitivity Tests ; Poultry ; *Salmonella enterica ; *Salmonella enteritidis/genetics ; }, abstract = {Several factors are involved in the emergence of antibiotic-resistant bacteria and pose a serious threat to public health safety. Among them, clustered regularly interspaced short palindromic repeat- (CRISPR-) Cas system, an adaptive immune system, is thought to be involved in the development of antibiotic resistance in bacteria. The current study was aimed at determining not only the presence of antibiotic resistance and CRISPR-Cas system but also their association with each other in Salmonella enteritidis isolated from the commercial poultry. A total of 139 samples were collected from poultry birds sold at the live bird markets of Lahore City, and both phenotypic and genotypic methods were used to determine antimicrobial resistance. The presence of the CRISPR-Cas system was determined by PCR, followed by sequencing. All isolates of S. enteritidis (100%) were resistant to nalidixic acid, whereas 95% of isolates were resistant to ampicillin. Five multidrug-resistant isolates (MDR) such as S. enteritidis isolate (S. E1, S. E2, S. E4, S. E5, and S. E8) were found in the present study. The CRISPR-Cas system was detected in all of these MDR isolates, and eight spacers were detected within the CRISPR array. In addition, an increased expression of CRISPR-related genes was observed in the standard strain and MDR S. enteritidis isolates. The association of the CRISPSR-Cas system with multiple drug resistance highlights the exogenous acquisition of genes by horizontal transfer. The information could be used further to combat antibiotic resistance in pathogens like Salmonella.}, }
@article {pmid35385916, year = {2022}, author = {Mallick, T and Mishra, R and Mohanty, S and Joshi, RK}, title = {Genome Wide Analysis of the Potato Soft Rot Pathogen Pectobacterium carotovorum Strain ICMP 5702 to Predict Novel Insights into Its Genetic Features.}, journal = {The plant pathology journal}, volume = {38}, number = {2}, pages = {102-114}, doi = {10.5423/PPJ.OA.12.2021.0190}, pmid = {35385916}, issn = {1598-2254}, support = {BT/PR23412/BPA/118/284/2017//Dept. of Biotechnology/ ; EMR/2016/005234//Science and Engineering Research Board/ ; //Dept. of Science and Technology/ ; }, abstract = {Pectobacterium carotovorum subsp. carotovorum (Pcc) is a gram-negative, broad host range bacterial pathogen which causes soft rot disease in potatoes as well as other vegetables worldwide. While Pectobacterium infection relies on the production of major cell wall degrading enzymes, other virulence factors and the mechanism of genetic adaptation of this pathogen is not yet clear. In the present study, we have performed an in-depth genome-wide characterization of Pcc strain ICMP5702 isolated from potato and compared it with other pathogenic bacteria from the Pectobacterium genus to identify key virulent determinants. The draft genome of Pcc ICMP5702 contains 4,774,457 bp with a G + C content of 51.90% and 4,520 open reading frames. Genome annotation revealed prominent genes encoding key virulence factors such as plant cell wall degrading enzymes, flagella-based motility, phage proteins, cell membrane structures, and secretion systems. Whereas, a majority of determinants were conserved among the Pectobacterium strains, few notable genes encoding AvrE-family type III secretion system effectors, pectate lyase and metalloprotease in addition to the CRISPR-Cas based adaptive immune system were uniquely represented. Overall, the information generated through this study will contribute to decipher the mechanism of infection and adaptive immunity in Pcc.}, }
@article {pmid35385325, year = {2022}, author = {Ahmed, ASI and Sheng, MHC and Lau, KW and Wilson, SM and Wongwarawat, D and Tang, X and Ghahramanpouri, M and Nehme, A and Xu, Y and Abdipour, A and Zhang, XB and Wasnik, S and Baylink, DJ}, title = {Calcium released by osteoclastic resorption stimulates autocrine/paracrine activities in local osteogenic cells to promote coupled bone formation.}, journal = {American journal of physiology. Cell physiology}, volume = {322}, number = {5}, pages = {C977-C990}, doi = {10.1152/ajpcell.00413.2021}, pmid = {35385325}, issn = {1522-1563}, support = {W81XWH-12-1-0023//DOD | US Army | MEDCOM | Telemedicine and Advanced Technology Research Center (TATRC)/ ; }, mesh = {Animals ; *Bone Resorption/metabolism ; Calcium/metabolism ; Calcium Channels/genetics/metabolism ; Cell Differentiation ; Mice ; Osteoclasts/metabolism ; *Osteogenesis ; RANK Ligand/metabolism ; Receptors, Calcium-Sensing/genetics/metabolism ; Vascular Endothelial Growth Factor A/metabolism ; }, abstract = {A major cause of osteoporosis is impaired coupled bone formation. Mechanistically, both osteoclast-derived and bone-derived growth factors have been previously implicated. Here, we hypothesize that the release of bone calcium during osteoclastic bone resorption is essential for coupled bone formation. Osteoclastic resorption increases interstitial fluid calcium locally from the normal 1.8 mM up to 5 mM. MC3T3-E1 osteoprogenitor cells, cultured in a 3.6 mM calcium medium, demonstrated that calcium signaling stimulated osteogenic cell proliferation, differentiation, and migration. Calcium channel knockdown studies implicated calcium channels, Cav1.2, store-operated calcium entry (SOCE), and calcium-sensing receptor (CaSR) in regulating bone cell anabolic activities. MC3T3-E1 cells cultured in a 3.6 mM calcium medium expressed increased gene expression of Wnt signaling and growth factors platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and bone morphogenic protein-2 (BMP 2). Our coupling model of bone formation, the receptor activator of nuclear factor-κΒ ligand (RANKL)-treated mouse calvaria, confirmed the role of calcium signaling in coupled bone formation by exhibiting increased gene expression for osterix and osteocalcin. Critically, dual immunocytochemistry showed that RANKL treatment increased osterix-positive cells and increased fluorescence intensity of Cav1.2 and CaSR protein expression per osterix-positive cell. The above data established that calcium released by osteoclasts contributed to the regulation of coupled bone formation. CRISPR/Cas-9 knockout of Cav1.2 in osteoprogenitor cells cultured in basal calcium medium caused a >80% decrease in the expression of downstream osteogenic genes, emphasizing the large magnitude of the effect of calcium signaling. Thus, calcium signaling is a major regulator of coupled bone formation.}, }
@article {pmid35385207, year = {2022}, author = {Yang, S and Joesaar, A and Bögels, BWA and Mann, S and de Greef, TFA}, title = {Protocellular CRISPR/Cas-Based Diffusive Communication Using Transcriptional RNA Signaling.}, journal = {Angewandte Chemie (International ed. in English)}, volume = {}, number = {}, pages = {e202202436}, doi = {10.1002/anie.202202436}, pmid = {35385207}, issn = {1521-3773}, support = {101000199/ERC_/European Research Council/International ; }, abstract = {Protocells containing enzyme-driven biomolecular circuits that can process and exchange information offer a promising approach for mimicking cellular features and developing molecular information platforms. Here, we employ synthetic transcriptional circuits together with CRISPR/Cas-based DNA processing inside semipermeable protein-polymer microcompartments. We first establish a transcriptional protocell that can be activated by external DNA strands and produce functional RNA aptamers. Subsequently, we engineer a transcriptional module to generate RNA strands functioning as diffusive signals that can be sensed by neighboring protocells and trigger the activation of internalized DNA probes or localization of Cas nucleases. Our results highlight the opportunities to combine CRISPR/Cas machinery and DNA nanotechnology for protocellular communication and provide a step towards the development of protocells capable of distributed molecular information processing.}, }
@article {pmid35384405, year = {2022}, author = {Koch, PJ and Webb, S and Gugger, JA and Salois, MN and Koster, MI}, title = {Differentiation of Human Induced Pluripotent Stem Cells into Keratinocytes.}, journal = {Current protocols}, volume = {2}, number = {4}, pages = {e408}, pmid = {35384405}, issn = {2691-1299}, support = {//National Foundation for Ectodermal Dysplasias/ ; R21EY029081/NH/NIH HHS/United States ; R21 EY029081/EY/NEI NIH HHS/United States ; R01 AR072621/AR/NIAMS NIH HHS/United States ; R01AR072621/NH/NIH HHS/United States ; }, mesh = {Cell Culture Techniques/methods ; Cell Differentiation/genetics ; Humans ; *Induced Pluripotent Stem Cells ; Keratinocytes ; Skin ; }, abstract = {Investigating basic biological mechanisms underlying human diseases relies on the availability of sufficient quantities of patient cells. As most primary somatic cells have a limited lifespan, obtaining sufficient material for biological studies has been a challenge. The development of induced pluripotent stem cell (iPSC) technology has been a game changer, especially in the field of rare genetic disorders. iPSC are essentially immortal, can be stored indefinitely, and can thus be used to generate defined somatic cells in unlimited quantities. Further, the availability of genome editing technologies, such as CRISPR/CAS, has provided us with the opportunity to create "designer" iPSC lines with defined genetic characteristics. A major advancement in biological research stems from the development of methods to direct iPSC differentiation into defined cell types. In this article, we provide the basic protocol for the generation of human iPSC-derived keratinocytes (iPSC-K). These cells have the characteristics of basal epidermal keratinocytes and represent a tool for the investigation of normal epidermal biology, as well as genetic and acquired skin disorders. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Directed differentiation of human iPSC into keratinocytes Support Protocol 1: Coating cell culture dishes or plates with Vitronectin XF™ Support Protocol 2: Freezing iPSC Support Protocol 3: Preparing AggreWell™ 400 6-well plates for EB formation Support Protocol 4: Coating cell culture dishes or plates with Collagen IV Support Protocol 5: Immunofluorescence staining of cells.}, }
@article {pmid35384352, year = {2022}, author = {Whitley, JA and Kim, S and Lou, L and Ye, C and Alsaidan, OA and Sulejmani, E and Cai, J and Desrochers, EG and Beharry, Z and Rickman, CB and Klingeborn, M and Liu, Y and Xie, ZR and Cai, H}, title = {Encapsulating Cas9 into extracellular vesicles by protein myristoylation.}, journal = {Journal of extracellular vesicles}, volume = {11}, number = {4}, pages = {e12196}, pmid = {35384352}, issn = {2001-3078}, support = {R21 AI157831/AI/NIAID NIH HHS/United States ; U01 CA225784/CA/NCI NIH HHS/United States ; R01 EY031748/EY/NEI NIH HHS/United States ; R21 EY028671/EY/NEI NIH HHS/United States ; P30 EY005722/EY/NEI NIH HHS/United States ; }, mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; *Extracellular Vesicles ; Gene Editing ; Genetic Therapy ; }, abstract = {CRISPR/Cas9 genome editing is a very promising avenue for the treatment of a variety of genetic diseases. However, it is still very challenging to encapsulate CRISPR/Cas9 machinery for delivery. Protein N-myristoylation is an irreversible co/post-translational modification that results in the covalent attachment of the myristoyl-group to the N-terminus of a target protein. It serves as an anchor for a protein to associate with the cell membrane and determines its intracellular trafficking and activity. Extracellular vesicles (EVs) are secreted vesicles that mediate cell-cell communication. In this study, we demonstrate that myristoylated proteins were preferentially encapsulated into EVs. The octapeptide derived from the leading sequence of the N-terminus of Src kinase was a favourable substrate for N-myristoyltransferase 1, the enzyme that catalyzes myristoylation. The fusion of the octapeptide onto the N-terminus of Cas9 promoted the myristoylation and encapsulation of Cas9 into EVs. Encapsulation of Cas9 and sgRNA-eGFP inside EVs was confirmed using protease digestion assays. Additionally, to increase the transfection potential, VSV-G was introduced into the EVs. The encapsulated Cas9 in EVs accounted for 0.7% of total EV protein. Importantly, the EVs coated with VSV-G encapsulating Cas9/sgRNA-eGFP showed up to 42% eGFP knock out efficiency with limited off-target effects in recipient cells. Our study provides a novel approach to encapsulate CRISPR/Cas9 protein and sgRNA into EVs. This strategy may open an effective avenue to utilize EVs as vehicles to deliver CRISPR/Cas9 for genome-editing-based gene therapy.}, }
@article {pmid35382990, year = {2022}, author = {Horie, M and Yamano-Adachi, N and Kawabe, Y and Kaneoka, H and Fujita, H and Nagamori, E and Iwai, R and Sato, Y and Kanie, K and Ohta, S and Somiya, M and Ino, K}, title = {Recent advances in animal cell technologies for industrial and medical applications.}, journal = {Journal of bioscience and bioengineering}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.jbiosc.2022.03.005}, pmid = {35382990}, issn = {1347-4421}, abstract = {The industrial use of living organisms for bioproduction of valued substances has been accomplished mostly using microorganisms. To produce high-value bioproducts such as antibodies that require glycosylation modification for better performance, animal cells have been recently gaining attention in bioengineering because microorganisms are unsuitable for producing such substances. Furthermore, animal cells are now classified as products because a large number of cells are required for use in regenerative medicine. In this article, we review animal cell technologies and the use of animal cells, focusing on useable cell generation and large-scale production of animal cells. We review recent advance in mammalian cell line development because this is the first step in the production of recombinant proteins, and it largely affects the efficacy of the production. We next review genetic engineering technology focusing on CRISPR-Cas system as well as surrounding technologies as these methods have been gaining increasing attention in areas that use animal cells. We further review technologies relating to bioreactors used in the context of animal cells because they are essential for the mass production of target products. We also review tissue engineering technology because tissue engineering is one of the main exits for mass-produced cells; in combination with genetic engineering technology, it can prove to be a promising treatment for patients with genetic diseases after the establishment of induced pluripotent stem cell technology. The technologies highlighted in this review cover brief outline of the recent animal cell technologies related to industrial and medical applications.}, }
@article {pmid35380812, year = {2022}, author = {Pan, R and Liu, J and Wang, P and Wu, D and Chen, J and Wu, Y and Li, G}, title = {Ultrasensitive CRISPR/Cas12a-Driven SERS Biosensor for On-Site Nucleic Acid Detection and Its Application to Milk Authenticity Testing.}, journal = {Journal of agricultural and food chemistry}, volume = {70}, number = {14}, pages = {4484-4491}, doi = {10.1021/acs.jafc.1c08262}, pmid = {35380812}, issn = {1520-5118}, mesh = {Alkalies ; Animals ; *Biosensing Techniques ; CRISPR-Cas Systems ; DNA/genetics ; Gold ; *Metal Nanoparticles ; Milk ; *Nucleic Acids ; Spectrum Analysis, Raman ; }, abstract = {An ultrasensitive surface-enhanced Raman scattering (SERS) biosensor driven by CRISPR/Cas12a was proposed for on-site nucleic acid detection. We tactfully modified single-strand DNA (ssDNA) with a target-responsive Prussian blue (PB) nanolabel to form a probe and fastened it in the microplate. Attributed to the specific base pairing and highly efficient trans-cleavage ability of the CRISPR/Cas12a effector, precise target DNA recognition and signal amplification can be achieved, respectively. In the presence of target DNA, trans-cleavage towards the probe was activated, leading to the release of a certain number of PB nanoparticles (NPs). Then, these free PB NPs would be removed. Under alkali treatment, the breakdown of the remaining PB NPs in the microplate was triggered, producing massive ferricyanide anions (Fe(CN)64-), which could exhibit a unique characteristic Raman peak that was located in the "biological Raman-silent region". By mixing the alkali-treated solution with the SERS substrate, Au@Ag core-shell NP, the concentration of the target DNA was finally exhibited as SERS signals with undisturbed background, which can be detected by a portable Raman spectrometer. Importantly, this strategy could display an ultralow detection limit of 224 aM for target DNA. Furthermore, by targeting cow milk as the adulterated ingredient in goat milk, the proposed biosensor was successfully applied to milk authenticity detection.}, }
@article {pmid35378361, year = {2022}, author = {Krueger, LA and Morris, AC}, title = {Generation of a zebrafish knock-in line expressing MYC-tagged Sox11a using CRISPR/Cas9 genome editing.}, journal = {Biochemical and biophysical research communications}, volume = {608}, number = {}, pages = {8-13}, pmid = {35378361}, issn = {1090-2104}, support = {F30 EY031545/EY/NEI NIH HHS/United States ; R01 EY021769/EY/NEI NIH HHS/United States ; TL1 TR001997/TR/NCATS NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems ; *Gene Editing/methods ; Gene Knock-In Techniques ; Recombinational DNA Repair ; *Zebrafish/genetics ; }, abstract = {Advances in CRISPR-Cas9 genome editing technology have strengthened the role of zebrafish as a model organism for genetics and developmental biology. These tools have led to a significant increase in the production of loss-of-function mutant zebrafish lines. However, the generation of precisely edited knock-in lines has remained a significant challenge in the field due to the decreased efficiency of homology directed repair (HDR). In this study, we overcame some of these challenges by combining available design tools and synthetic, commercially available CRISPR reagents to generate a knock-in line carrying an in-frame MYC epitope tag at the sox11a locus. Zebrafish Sox11a is a transcription factor with critical roles in organogenesis, neurogenesis, craniofacial, and skeletal development; however, only a few direct molecular targets of Sox11a have been identified. Here, we evaluate the knock-in efficiency of various HDR donor configurations and demonstrate the successful expression and localization of the resulting knock-in allele. Our results provide an efficient, streamlined approach to knock-in experiments in zebrafish, which will enable expansion of downstream experimental applications that have previously been difficult to perform. Moreover, the MYC-Sox11a line we have generated will allow further investigation into the function and direct targets of Sox11a.}, }
@article {pmid35377968, year = {2022}, author = {Cheng, Y and Sretenovic, S and Zhang, Y and Pan, C and Huang, J and Qi, Y}, title = {Expanding the targeting scope of FokI-dCas nuclease systems with SpRY and Mb2Cas12a.}, journal = {Biotechnology journal}, volume = {}, number = {}, pages = {e2100571}, doi = {10.1002/biot.202100571}, pmid = {35377968}, issn = {1860-7314}, abstract = {CRISPR-Cas9 and Cas12a are widely used sequence-specific nucleases (SSNs) for genome editing. The nuclease domains of Cas proteins can induce DNA double strand breaks upon RNA guided DNA targeting. Zinc finger nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) have been popular SSNs prior to CRISPR. Both ZFNs and TALENs are based on reconstitution of two monomers with each consisting of a DNA binding domain and a FokI nuclease domain. Inspired by the configuration of ZFNs and TALENs, dimeric FokI-dCas9 systems were previously demonstrated in human cells. Such configuration, based on a pair of guide RNAs (gRNAs), offers great improvement on targeting specificity. To expand the targeting scope of dimeric FokI-dCas systems, the PAM (protospacer adjacent motif)-less SpRY Cas9 variant and the PAM-relaxed Mb2Cas12a system were explored. Rice cells showed that FokI-dSpRY had more robust editing efficiency than a paired SpRY nickase system. Furthermore, a dimeric FokI-dMb2Cas12a system was developed that displayed comparable editing activity to Mb2Cas12a nuclease in rice cells. Finally, a single-chain FokI-FokI-dMb2Cas12a system was developed that cuts DNA outside its targeting sequence, which could be useful for many versatile applications. Together, this work greatly expanded the FokI based CRISPR-Cas systems for genome editing.}, }
@article {pmid35377365, year = {2022}, author = {Yan, Q and He, Y and Yue, Y and Jie, L and Wen, T and Zhao, Y and Zhang, M and Zhang, T}, title = {Construction of Homozygous Mutants of Migratory Locust using CRISPR/Cas9 Technology.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {181}, pages = {}, doi = {10.3791/63629}, pmid = {35377365}, issn = {1940-087X}, mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/methods ; *Grasshoppers/genetics/metabolism ; Humans ; RNA, Guide/genetics ; Technology ; }, abstract = {The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture, preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.}, }
@article {pmid35373735, year = {2022}, author = {Cornean, A and Gierten, J and Welz, B and Mateo, JL and Thumberger, T and Wittbrodt, J}, title = {Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction.}, journal = {eLife}, volume = {11}, number = {}, pages = {}, pmid = {35373735}, issn = {2050-084X}, support = {WI 1824/9-1//Deutsche Forschungsgemeinschaft/ ; 810172//H2020 European Research Council/ ; S/02/17//Deutsche Herzstiftung/ ; 3DMM2O//Deutsche Forschungsgemeinschaft/ ; 81X2500189//Deutsches Zentrum für Herz-Kreislaufforschung/ ; Add-on Fellowship//Joachim Herz Stiftung/ ; 3DMM2O, EXC 2082/1 Wittbrodt C3//Deutsche Forschungsgemeinschaft/ ; }, mesh = {Adenine ; Animals ; CRISPR-Cas Systems ; Cytosine ; DNA ; *Gene Editing ; Mutation ; *Zebrafish/genetics ; }, abstract = {Single nucleotide variants (SNVs) are prevalent genetic factors shaping individual trait profiles and disease susceptibility. The recent development and optimizations of base editors, rubber and pencil genome editing tools now promise to enable direct functional assessment of SNVs in model organisms. However, the lack of bioinformatic tools aiding target prediction limits the application of base editing in vivo. Here, we provide a framework for adenine and cytosine base editing in medaka (Oryzias latipes) and zebrafish (Danio rerio), ideal for scalable validation studies. We developed an online base editing tool ACEofBASEs (a careful evaluation of base-edits), to facilitate decision-making by streamlining sgRNA design and performing off-target evaluation. We used state-of-the-art adenine (ABE) and cytosine base editors (CBE) in medaka and zebrafish to edit eye pigmentation genes and transgenic GFP function with high efficiencies. Base editing in the genes encoding troponin T and the potassium channel ERG faithfully recreated known cardiac phenotypes. Deep-sequencing of alleles revealed the abundance of intended edits in comparison to low levels of insertion or deletion (indel) events for ABE8e and evoBE4max. We finally validated missense mutations in novel candidate genes of congenital heart disease (CHD) dapk3, ube2b, usp44, and ptpn11 in F0 and F1 for a subset of these target genes with genotype-phenotype correlation. This base editing framework applies to a wide range of SNV-susceptible traits accessible in fish, facilitating straight-forward candidate validation and prioritization for detailed mechanistic downstream studies.}, }
@article {pmid35373270, year = {2022}, author = {}, title = {A Base Editing Platform Shows Function of Cancer-Associated Variants.}, journal = {Cancer discovery}, volume = {12}, number = {4}, pages = {883}, doi = {10.1158/2159-8290.CD-RW2022-034}, pmid = {35373270}, issn = {2159-8290}, mesh = {CRISPR-Cas Systems ; *Gene Editing ; Humans ; *Neoplasms/genetics/therapy ; }, abstract = {A resource was developed to introduce and interrogate cancer-associated single-nucleotide variants.}, }
@article {pmid35373187, year = {2022}, author = {Ravendran, S and Hernández, SS and König, S and Bak, RO}, title = {CRISPR/Cas-Based Gene Editing Strategies for DOCK8 Immunodeficiency Syndrome.}, journal = {Frontiers in genome editing}, volume = {4}, number = {}, pages = {793010}, pmid = {35373187}, issn = {2673-3439}, abstract = {Defects in the DOCK8 gene causes combined immunodeficiency termed DOCK8 immunodeficiency syndrome (DIDS). DIDS previously belonged to the disease category of autosomal recessive hyper IgE syndrome (AR-HIES) but is now classified as a combined immunodeficiency (CID). This genetic disorder induces early onset of susceptibility to severe recurrent viral and bacterial infections, atopic diseases and malignancy resulting in high morbidity and mortality. This pathological state arises from impairment of actin polymerization and cytoskeletal rearrangement, which induces improper immune cell migration-, survival-, and effector functions. Owing to the severity of the disease, early allogenic hematopoietic stem cell transplantation is recommended even though it is associated with risk of unintended adverse effects, the need for compatible donors, and high expenses. So far, no alternative therapies have been developed, but the monogenic recessive nature of the disease suggests that gene therapy may be applied. The advent of the CRISPR/Cas gene editing system heralds a new era of possibilities in precision gene therapy, and positive results from clinical trials have already suggested that the tool may provide definitive cures for several genetic disorders. Here, we discuss the potential application of different CRISPR/Cas-mediated genetic therapies to correct the DOCK8 gene. Our findings encourage the pursuit of CRISPR/Cas-based gene editing approaches, which may constitute more precise, affordable, and low-risk definitive treatment options for DOCK8 deficiency.}, }
@article {pmid35371164, year = {2022}, author = {Ali, Q and Yu, C and Hussain, A and Ali, M and Ahmar, S and Sohail, MA and Riaz, M and Ashraf, MF and Abdalmegeed, D and Wang, X and Imran, M and Manghwar, H and Zhou, L}, title = {Genome Engineering Technology for Durable Disease Resistance: Recent Progress and Future Outlooks for Sustainable Agriculture.}, journal = {Frontiers in plant science}, volume = {13}, number = {}, pages = {860281}, pmid = {35371164}, issn = {1664-462X}, abstract = {Crop production worldwide is under pressure from multiple factors, including reductions in available arable land and sources of water, along with the emergence of new pathogens and development of resistance in pre-existing pathogens. In addition, the ever-growing world population has increased the demand for food, which is predicted to increase by more than 100% by 2050. To meet these needs, different techniques have been deployed to produce new cultivars with novel heritable mutations. Although traditional breeding continues to play a vital role in crop improvement, it typically involves long and laborious artificial planting over multiple generations. Recently, the application of innovative genome engineering techniques, particularly CRISPR-Cas9-based systems, has opened up new avenues that offer the prospects of sustainable farming in the modern agricultural industry. In addition, the emergence of novel editing systems has enabled the development of transgene-free non-genetically modified plants, which represent a suitable option for improving desired traits in a range of crop plants. To date, a number of disease-resistant crops have been produced using gene-editing tools, which can make a significant contribution to overcoming disease-related problems. Not only does this directly minimize yield losses but also reduces the reliance on pesticide application, thereby enhancing crop productivity that can meet the globally increasing demand for food. In this review, we describe recent progress in genome engineering techniques, particularly CRISPR-Cas9 systems, in development of disease-resistant crop plants. In addition, we describe the role of CRISPR-Cas9-mediated genome editing in sustainable agriculture.}, }
@article {pmid35369445, year = {2022}, author = {Santana de Carvalho, D and Trovatti Uetanabaro, AP and Kato, RB and Aburjaile, FF and Jaiswal, AK and Profeta, R and De Oliveira Carvalho, RD and Tiwar, S and Cybelle Pinto Gomide, A and Almeida Costa, E and Kukharenko, O and Orlovska, I and Podolich, O and Reva, O and Ramos, PIP and De Carvalho Azevedo, VA and Brenig, B and Andrade, BS and de Vera, JP and Kozyrovska, NO and Barh, D and Góes-Neto, A}, title = {The Space-Exposed Kombucha Microbial Community Member Komagataeibacter oboediens Showed Only Minor Changes in Its Genome After Reactivation on Earth.}, journal = {Frontiers in microbiology}, volume = {13}, number = {}, pages = {782175}, pmid = {35369445}, issn = {1664-302X}, abstract = {Komagataeibacter is the dominant taxon and cellulose-producing bacteria in the Kombucha Microbial Community (KMC). This is the first study to isolate the K. oboediens genome from a reactivated space-exposed KMC sample and comprehensively characterize it. The space-exposed genome was compared with the Earth-based reference genome to understand the genome stability of K. oboediens under extraterrestrial conditions during a long time. Our results suggest that the genomes of K. oboediens IMBG180 (ground sample) and K. oboediens IMBG185 (space-exposed) are remarkably similar in topology, genomic islands, transposases, prion-like proteins, and number of plasmids and CRISPR-Cas cassettes. Nonetheless, there was a difference in the length of plasmids and the location of cas genes. A small difference was observed in the number of protein coding genes. Despite these differences, they do not affect any genetic metabolic profile of the cellulose synthesis, nitrogen-fixation, hopanoid lipids biosynthesis, and stress-related pathways. Minor changes are only observed in central carbohydrate and energy metabolism pathways gene numbers or sequence completeness. Altogether, these findings suggest that K. oboediens maintains its genome stability and functionality in KMC exposed to the space environment most probably due to the protective role of the KMC biofilm. Furthermore, due to its unaffected metabolic pathways, this bacterial species may also retain some promising potential for space applications.}, }
@article {pmid35369433, year = {2022}, author = {Riedl, A and Fischer, J and Burgert, HG and Ruzsics, Z}, title = {Rescue of Recombinant Adenoviruses by CRISPR/Cas-Mediated in vivo Terminal Resolution.}, journal = {Frontiers in microbiology}, volume = {13}, number = {}, pages = {854690}, pmid = {35369433}, issn = {1664-302X}, abstract = {Recombinant adenovirus (rAd) vectors represent one of the most frequently used vehicles for gene transfer applications in vitro and in vivo. rAd genomes are constructed in Escherichia coli where their genomes can be maintained, propagated, and modified in form of circular plasmids or bacterial artificial chromosomes. Although the rescue of rAds from their circular plasmid or bacmid forms is well established, it works with relatively low primary efficiency, preventing this technology for library applications. To overcome this barrier, we tested a novel strategy for the reconstitution of rAds that utilizes the CRISPR/Cas-machinery to cleave the circular rAd genomes in close proximity to their inverted terminal repeats (ITRs) within the producer cells upon transfection. This CRISPR/Cas-mediated in vivo terminal resolution allowed efficient rescue of vectors derived from different human adenovirus (HAdV) species. By this means, it was not only possible to increase the efficiency of virus rescue by about 50-fold, but the presented methodology appeared also remarkably simpler and faster than traditional rAd reconstitution methods.}, }
@article {pmid35368966, year = {2022}, author = {Cai, J and Wu, D and Jin, Y and Bao, S}, title = {Effect of CMB Carrying PTX and CRISPR/Cas9 on Endometrial Cancer Naked Mouse Model.}, journal = {Journal of healthcare engineering}, volume = {2022}, number = {}, pages = {7119195}, pmid = {35368966}, issn = {2040-2309}, mesh = {Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; *Endometrial Neoplasms/genetics ; Female ; Glycogen Synthase Kinase 3 ; Humans ; Mammals ; Mice ; *Paclitaxel/pharmacology/therapeutic use ; }, abstract = {Endometrial cancer, one of the most common gynecological cancers in women. Patients with advanced or recurrent disease have poor long-term outcomes. The current experiment explore the roles of cationic microbubbles (CMBs) carrying paclitaxel (PTX) and CRISPR/Cas9 plasmids on the xenotransplantation model of mice with endometrial cancer. The tumor histology, tumor cell viability, cell cycle, and invasion ability were investigated. Meanwhile, the P27, P21, GSK-3, Bcl-2 associated death promoter (Bad), mammalian target of rapamycin (mTOR), and C-erbB-2 expressions were evaluated by qRT-PCR and western blotting, respectively. CMB-PTX-CRISPR/Cas9 had an inhibitory action on the tumor growth, tumor cell viability, cell cycle, and invasion ability of the mouse xenograft model of endometrial cancer. The CMB-PTX-CRISPR/Cas9 increased the GSK-3, P21, P27, and Bad expression levels, while reduced the C-erbB-2 and mTOR expressions. CMBs loaded with both PTX and CRISPR/Cas9 plasmids may be a new combination treatment with much potential. CMB-PTX-CRISPR/Cas9 may regulate the tumor cell viability, invasion, and metastasis of endometrial cancer naked mouse model by upregulating expressions of GSK-3, P21, P27, and Bad.}, }
@article {pmid35366394, year = {2022}, author = {Kurihara, N and Nakagawa, R and Hirano, H and Okazaki, S and Tomita, A and Kobayashi, K and Kusakizako, T and Nishizawa, T and Yamashita, K and Scott, DA and Nishimasu, H and Nureki, O}, title = {Structure of the type V-C CRISPR-Cas effector enzyme.}, journal = {Molecular cell}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.molcel.2022.03.006}, pmid = {35366394}, issn = {1097-4164}, abstract = {RNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Recent studies identified functionally divergent type V Cas12 family enzymes. Among them, Cas12c2 binds a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) and recognizes double-stranded DNA targets with a short TN PAM. Here, we report the cryo-electron microscopy structures of the Cas12c2-guide RNA binary complex and the Cas12c2-guide RNA-target DNA ternary complex. The structures revealed that the crRNA and tracrRNA form an unexpected X-junction architecture, and that Cas12c2 recognizes a single T nucleotide in the PAM through specific hydrogen-bonding interactions with two arginine residues. Furthermore, our biochemical analyses indicated that Cas12c2 processes its precursor crRNA to a mature crRNA using the RuvC catalytic site through a unique mechanism. Collectively, our findings improve the mechanistic understanding of diverse type V CRISPR-Cas effectors.}, }
@article {pmid35366350, year = {2022}, author = {Murugesan, AC and Varughese, HS}, title = {Analysis of CRISPR-Cas system and antimicrobial resistance in Staphylococcus coagulans isolates.}, journal = {Letters in applied microbiology}, volume = {}, number = {}, pages = {}, doi = {10.1111/lam.13713}, pmid = {35366350}, issn = {1472-765X}, support = {EMR/2016/006141//Department of Science and Technology-Science and Engineering Research Board, Government of India/ ; }, abstract = {CRISPR-Cas system contributes adaptive immunity to protect the bacterial and archaeal genome against invading mobile genetic elements. In this study, an attempt was made to characterize the CRISPR-Cas system in Staphylococcus coagulans, the second most prevalent coagulase positive staphylococci causing skin infections in dogs. Out of 45 S. coagulans isolates, 42/45 (93·33%) strains contained CRISPR-Cas system and 45 confirmed CRISPR system was identified in 42 S. coagulans isolates. The length of CRISPR loci ranged from 167 to 2477 bp, and the number of spacers in each CRISPR was varied from two spacers to as high as 37 numbers. Direct repeat (DR) sequences were between 30 and 37, but most (35/45) of the DRs contained 36 sequences. The predominant S. coagulans strains 29/45 did not possess any antimicrobial resistant genes (ARG); 26/29 strains contained Type IIC CRISPR-Cas system. Three isolates from Antarctica seals neither contain CRISPR-Cas system nor ARG. Only 15/45 S. coagulans strains (33·33%) harboured at least one ARG and 13/15 of them were having mecA gene. All the methicillin susceptible S. coagulans isolates contained Type IIC CRISPR-Cas system. In contrast, many (10/13) S. coagulans isolates which were methicillin resistant had Type IIIA CRISPR-Cas system, and this Type IIIA CRISPR-Cas system was present within the SCCmec mobile genetic element. Hence, this study suggests that Type II CRISPR-Cas in S. coagulans isolates might have played a possible role in preventing acquisition of plasmid/phage invasion and Type IIIA CRISPR-Cas system may have an insignificant role in the prevention of horizontal gene transfer of antimicrobial resistance genes in S. coagulans species.}, }
@article {pmid35365834, year = {2022}, author = {Poudel, R and Rodriguez, LT and Reisch, CR and Rivers, AR}, title = {GuideMaker: Software to design CRISPR-Cas guide RNA pools in non-model genomes.}, journal = {GigaScience}, volume = {11}, number = {}, pages = {}, pmid = {35365834}, issn = {2047-217X}, support = {//U.S. Department of Agriculture/ ; 6066-21310-005-D//American Radium Society/ ; //University of Florida/ ; }, mesh = {*CRISPR-Cas Systems ; Gene Editing ; Genome ; *RNA, Guide/genetics ; Software ; }, abstract = {BACKGROUND: CRISPR-Cas systems have expanded the possibilities for gene editing in bacteria and eukaryotes. There are many excellent tools for designing CRISPR-Cas guide RNAs (gRNAs) for model organisms with standard Cas enzymes. GuideMaker is intended as a fast and easy-to-use design tool for challenging projects with (i) non-standard Cas enzymes, (ii) non-model organisms, or (iii) projects that need to design a panel of gRNA for genome-wide screens.<br><br>FINDINGS: GuideMaker can rapidly design gRNAs for gene targets across the genome using a degenerate protospacer-adjacent motif (PAM) and a genome. The tool applies hierarchical navigable small world graphs to speed up the comparison of guide RNAs and optionally provides on-target and off-target scoring. This allows the user to design effective gRNAs targeting all genes in a typical bacterial genome in ∼1-2 minutes.<br><br>CONCLUSIONS: GuideMaker enables the rapid design of genome-wide gRNA for any CRISPR-Cas enzyme in non-model organisms. While GuideMaker is designed with prokaryotic genomes in mind, it can efficiently process eukaryotic genomes as well. GuideMaker is available as command-line software, a stand-alone web application, and a tool in the CyCverse Discovery Environment. All versions are available under a Creative Commons CC0 1.0 Universal Public Domain Dedication.}, }
@article {pmid35364533, year = {2022}, author = {Lin, X and Li, C and Meng, X and Yu, W and Duan, N and Wang, Z and Wu, S}, title = {CRISPR-Cas12a-mediated luminescence resonance energy transfer aptasensing platform for deoxynivalenol using gold nanoparticle-decorated Ti3C2Tx MXene as the enhanced quencher.}, journal = {Journal of hazardous materials}, volume = {433}, number = {}, pages = {128750}, doi = {10.1016/j.jhazmat.2022.128750}, pmid = {35364533}, issn = {1873-3336}, mesh = {*Biosensing Techniques ; CRISPR-Cas Systems ; Fluorescence Resonance Energy Transfer/methods ; Gold ; Luminescence ; *Metal Nanoparticles ; Titanium ; Trichothecenes ; }, abstract = {Deoxynivalenol (DON) is a typical mycotoxin in cereals and poses tremendous threats to the ecological environment and public health. Therefore, exploiting sensitive and robust analytical methods for DON is particularly important. Here, we fabricated a CRISPR-Cas12a-mediated luminescence resonance energy transfer (LRET) aptasensor to detect DON by using single-stranded DNA modified upconversion nanoparticles (ssDNA-UCNPs) as anti-interference luminescence labels and gold nanoparticle-decorated Ti3C2Tx MXene nanosheets (MXene-Au) as enhanced quenchers. The DON aptamer can activate the trans-cleavage activity of Cas12a to indiscriminately cut nearby ssDNA-UCNPs into small fragments, which prevents ssDNA-UCNPs from adsorbing onto MXene-Au, and the upconversion luminescence (UCL) remains. Upon the binding of the aptamer with DON, the trans-cleavage activity of Cas12a was suppressed, and the ssDNA-UCNPs were not cleaved and easily adsorbed onto MXene-Au, which caused UCL quenching. Under optimized conditions, the limit of detection was determined to be 0.64 ng/mL with a linear range of 1 - 500 ng/mL. In addition, the sensor was successfully applied to detect DON in corn flour and Tai Lake water with recoveries of 96.2 - 105% and 95.2 - 104%, respectively. This platform achieves a sensitive and specific analysis of DON and greatly broadens the detection range of CRISPR-Cas sensors for non-nucleic acids hazards in the environment and food.}, }
@article {pmid35363475, year = {2022}, author = {Malcı, K and Walls, LE and Rios-Solis, L}, title = {Rational Design of CRISPR/Cas12a-RPA Based One-Pot COVID-19 Detection with Design of Experiments.}, journal = {ACS synthetic biology}, volume = {11}, number = {4}, pages = {1555-1567}, pmid = {35363475}, issn = {2161-5063}, mesh = {*COVID-19/diagnosis ; CRISPR-Cas Systems/genetics ; Humans ; Nucleic Acid Amplification Techniques/methods ; Pandemics ; RNA, Viral/analysis/genetics ; SARS-CoV-2/genetics ; Sensitivity and Specificity ; }, abstract = {Simple and effective molecular diagnostic methods have gained importance due to the devastating effects of the COVID-19 pandemic. Various isothermal one-pot COVID-19 detection methods have been proposed as favorable alternatives to standard RT-qPCR methods as they do not require sophisticated and/or expensive devices. However, as one-pot reactions are highly complex with a large number of variables, determining the optimum conditions to maximize sensitivity while minimizing diagnostic cost can be cumbersome. Here, statistical design of experiments (DoE) was employed to accelerate the development and optimization of a CRISPR/Cas12a-RPA-based one-pot detection method for the first time. Using a definitive screening design, factors with a significant effect on performance were elucidated and optimized, facilitating the detection of two copies/μL of full-length SARS-CoV-2 (COVID-19) genome using simple instrumentation. The screening revealed that the addition of a reverse transcription buffer and an RNase inhibitor, components generally omitted in one-pot reactions, improved performance significantly, and optimization of reverse transcription had a critical impact on the method's sensitivity. This strategic method was also applied in a second approach involving a DNA sequence of the N gene from the COVID-19 genome. The slight differences in optimal conditions for the methods using RNA and DNA templates highlight the importance of reaction-specific optimization in ensuring robust and efficient diagnostic performance. The proposed detection method is automation-compatible, rendering it suitable for high-throughput testing. This study demonstrated the benefits of DoE for the optimization of complex one-pot molecular diagnostics methods to increase detection sensitivity.}, }
@article {pmid35362932, year = {2022}, author = {Courtright-Lim, A}, title = {"CRISPR for Disabilities: How to Self-Regulate" or Something?.}, journal = {Journal of bioethical inquiry}, volume = {19}, number = {1}, pages = {151-161}, pmid = {35362932}, issn = {1176-7529}, mesh = {CRISPR-Cas Systems ; *Disabled Persons ; *Gene Editing ; Humans ; Stem Cell Research ; }, abstract = {The development of the CRISPR gene editing technique has been hyped as a technique that could fundamentally change scientific research and its clinical application. Unrecognized is the fact that it joins other technologies that have tried and failed under the same discourse of scientific hype. These technologies, like gene therapy and stem cell research, have moved quickly passed basic research into clinical application with dire consequences. Before hastily moving to clinical applications, it is necessary to consider basic research and determine how CRISPR/Cas systems should be applied. In the case of single gene diseases, that application is expected to have positive impacts, but as we shift to more complex diseases, the impact could be unintentionally negative. In the context of common disabilities, the level of genetic complexity may render this technology useless but potentially toxic, aggravating a social discourse that devalues those with disabilities. This paper intends to define the issues related to disability that are associated with using the CRIPSR/Cas system in basic research. It also aims to provide a decision tree to help determine whether the technology should be utilized or if alternative approaches beyond scientific research could lead to a better use of limited funding resources.}, }
@article {pmid35358830, year = {2022}, author = {Liu, Y and Jin, Y and Chen, T and Wu, Y and Peng, X and Li, W and Wei, S and Chen, M and Zou, Q and Guo, S and Xu, J and Tang, C and Zhou, X}, title = {Generation of a homozygous ARHGAP11B knockout hiPSC line by CRISPR/Cas9 system.}, journal = {Stem cell research}, volume = {61}, number = {}, pages = {102764}, doi = {10.1016/j.scr.2022.102764}, pmid = {35358830}, issn = {1876-7753}, mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; GTPase-Activating Proteins ; Gene Editing ; Homozygote ; Humans ; *Induced Pluripotent Stem Cells ; }, abstract = {The human specific gene ARHGAP11B is preferentially expressed in neural progenitors of fetal neocortex and plays a key role in the evolutionary expansion of the neocortex. Here, we generated a homozygous ARHGAP11B knockout human induced pluripotent stem cell (hiPSC) line through CRISPR/Cas9 gene editing system. ARHGAP11B deficient cell line maintained a normal karyotype (46, XX), expressed pluripotency markers, and showed the capability to spontaneously differentiate into all three germ layers in vivo. The ARHGAP11B knockout cell line can provide a new cell model for studying the evolution of human neocortex.}, }
@article {pmid35358611, year = {2022}, author = {Bernard, BE and Landmann, E and Jeker, LT and Schumann, K}, title = {CRISPR/Cas-based Human T cell Engineering: Basic Research and Clinical Application.}, journal = {Immunology letters}, volume = {245}, number = {}, pages = {18-28}, doi = {10.1016/j.imlet.2022.03.005}, pmid = {35358611}, issn = {1879-0542}, mesh = {*CRISPR-Cas Systems ; Cell Engineering ; *Gene Editing ; Genetic Engineering ; Humans ; T-Lymphocytes ; }, abstract = {Engineering human T cells for the treatment of cancer, viral infections and autoimmunity has been a long-standing dream of many immunologists and hematologists. Although primary human T cells have been genetically engineered for decades, this process was challenging, time consuming and mostly limited to transgene insertions mediated by viral transduction. The absence of widely accessible tools to efficiently and precisely engineer T cells genetically in a targeted manner limited their applicability as a living drug. This fundamentally changed with the discovery of CRISPR/Cas9 and its adaptation to human T cells. CRISPR/Cas9 has made T cell engineering widely accessible and accelerated the development of engineered adoptive T cell therapies. Only 6 years after the discovery of CRISPR/Cas9 as a biotechnological tool the first CRISPR engineered T cells have been administered to patients with refractory cancers in a phase I clinical trial. Novel Cas proteins - natural and engineered ones - are rapidly emerging. These offer for instance increased flexibility, activity and/or specificity. Moreover, sophisticated protein engineering and fusions of Cas with deaminases or reverse transcriptases enable genomic DNA editing without the need for a double strand cut. Thus, the "CRISPR tool box" for experimental use as well as for novel therapeutic approaches is rapidly expanding. In this review, we will summarize the current state of CRISPR/Cas-based engineering in human T cells for basic research and its clinical applications.}, }
@article {pmid35357193, year = {2022}, author = {Wu, T and Cao, Y and Liu, Q and Wu, X and Shang, Y and Piao, J and Li, Y and Dong, Y and Liu, D and Wang, H and Liu, J and Ding, B}, title = {Genetically Encoded Double-Stranded DNA-Based Nanostructure Folded by a Covalently Bivalent CRISPR/dCas System.}, journal = {Journal of the American Chemical Society}, volume = {144}, number = {14}, pages = {6575-6582}, doi = {10.1021/jacs.2c01760}, pmid = {35357193}, issn = {1520-5126}, mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics/metabolism ; Gene Editing ; *Nanostructures ; Ribonucleoproteins ; }, abstract = {DNA nanotechnology has been widely employed in the construction of various functional nanostructures. However, most DNA nanostructures rely on hybridization between multiple single-stranded DNAs. Herein, we report a general strategy for the construction of a double-stranded DNA-ribonucleoprotein (RNP) hybrid nanostructure by folding double-stranded DNA with a covalently bivalent clustered regularly interspaced short palindromic repeats (CRISPR)/nuclease-dead CRISPR-associated protein (dCas) system. In our design, dCas9 and dCas12a can be efficiently fused together through a flexible and stimuli-responsive peptide linker. After activation by guide RNAs, the covalently bivalent dCas9-12a RNPs (staples) can precisely recognize their target sequences in the double-stranded DNA scaffold and pull them together to construct a series of double-stranded DNA-RNP hybrid nanostructures. The genetically encoded hybrid nanostructure can protect genetic information in the folded state, similar to the natural DNA-protein hybrids present in chromosomes, and elicit efficient stimuli-responsive gene transcription in the unfolded form. This rationally developed double-stranded DNA folding and unfolding strategy presents a new avenue for the development of DNA nanotechnology.}, }
@article {pmid35355475, year = {2022}, author = {Zhou, Z and Lü, X and Zhu, L and Zhou, J and Huang, H and Zhang, C and Liu, X}, title = {[Construction of a stable TrxR1 knockout HCT-116 cell line using CRISPR/Cas9 gene editing system].}, journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology}, volume = {38}, number = {3}, pages = {1074-1085}, doi = {10.13345/j.cjb.210635}, pmid = {35355475}, issn = {1872-2075}, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Gene Knockout Techniques ; HCT116 Cells ; Humans ; RNA, Guide/genetics/metabolism ; }, abstract = {To investigate the cellular target selectivity of small molecules targeting thioredoxin reductase 1, we reported the construction and functional research of a stable TrxR1 gene (encode thioredoxin reductase 1) knockout HCT-116 cell line. We designed and selected TrxR1 knockout sites according to the TrxR1 gene sequence and CRISPR/Cas9 target designing principles. SgRNA oligos based on the selected TrxR1 knockout sites were obtained. Next, we constructed knockout plasmid by cloning the sgRNA into the pCasCMV-Puro-U6 vector. After transfection of the plasmid into HCT-116 cells, TrxR1 knockout HCT-116 cells were selected using puromycin resistance. The TrxR1 knockout efficiency was identified and verified by DNA sequencing, immunoblotting, TRFS-green fluorescent probe, and cellular TrxR1 enzyme activity detection. Finally, the correlation between TrxR1 expression and cellular effects of drugs specifically targeting TrxR1 was investigated by CCK-8 assay. The results demonstrated that the knockout plasmid expressing the sgRNA effectively knocked-out TrxR1 gene within HCT-116 cells, and no expression of TrxR1 protein could be observed in stable TrxR1 knockout HCT-116 (HCT116-TrxR1-KO) cells. The TrxR1-targeting inhibitor auranofin did not show any inhibitory activity against either cellular TrxR1 enzyme activity or cell proliferation. Based on these results, we conclude that a stable TrxR1 gene knockout HCT-116 cell line was obtained through CRISPR/Cas9 techniques, which may facilitate investigating the role of TrxR1 in various diseases.}, }
@article {pmid35354039, year = {2022}, author = {Yi, C and Cai, C and Cheng, Z and Zhao, Y and Yang, X and Wu, Y and Wang, X and Jin, Z and Xiang, Y and Jin, M and Han, L and Zhang, A}, title = {Genome-wide CRISPR-Cas9 screening identifies the CYTH2 host gene as a potential therapeutic target of influenza viral infection.}, journal = {Cell reports}, volume = {38}, number = {13}, pages = {110559}, doi = {10.1016/j.celrep.2022.110559}, pmid = {35354039}, issn = {2211-1247}, mesh = {Antiviral Agents/pharmacology ; CRISPR-Cas Systems/genetics ; Endosomes ; Humans ; *Influenza A virus/genetics ; *Influenza, Human/drug therapy/genetics ; }, abstract = {Host genes critical for viral infection are effective antiviral drug targets with tremendous potential due to their universal characteristics against different subtypes of viruses and minimization of drug resistance. Accordingly, we execute a genome-wide CRISPR-Cas9 screen with multiple rounds of survival selection. Enriched in this screen are several genes critical for host sialic acid biosynthesis and transportation, including the cytohesin 2 (CYTH2), tetratricopeptide repeat protein 24 (TTC24), and N-acetylneuraminate synthase (NANS), which we confirm are responsible for efficient influenza viral infection. Moreover, we reveal that CYTH2 is required for the early stage of influenza virus infection by mediating endosomal trafficking. Furthermore, CYTH2 antagonist SecinH3 blunts influenza virus infection in vivo. In summary, these data suggest that CYTH2 is an attractive target for developing host-directed antiviral drugs and therapeutics against influenza virus infection.}, }
@article {pmid35353638, year = {2022}, author = {Rallapalli, KL and Ranzau, BL and Ganapathy, KR and Paesani, F and Komor, AC}, title = {Combined Theoretical, Bioinformatic, and Biochemical Analyses of RNA Editing by Adenine Base Editors.}, journal = {The CRISPR journal}, volume = {5}, number = {2}, pages = {294-310}, doi = {10.1089/crispr.2021.0131}, pmid = {35353638}, issn = {2573-1602}, support = {R21 GM135736/GM/NIGMS NIH HHS/United States ; R35 GM138317/GM/NIGMS NIH HHS/United States ; T32 GM112584/GM/NIGMS NIH HHS/United States ; }, mesh = {Adenine/metabolism ; CRISPR-Cas Systems ; Computational Biology ; *Gene Editing ; RNA/genetics ; *RNA Editing/genetics ; }, abstract = {Adenine base editors (ABEs) have been subjected to multiple rounds of mutagenesis with the goal of optimizing their function as efficient and precise genome editing agents. Despite an ever-expanding data set of ABE mutants and their corresponding DNA or RNA-editing activity, the molecular mechanisms defining these changes remain to be elucidated. In this study, we provide a systematic interpretation of the nature of these mutations using an entropy-based classification model that relies on evolutionary data from extant protein sequences. Using this model in conjunction with experimental analyses, we identify two previously reported mutations that form an epistatic pair in the RNA-editing functional landscape of ABEs. Molecular dynamics simulations reveal the atomistic details of how these two mutations affect substrate-binding and catalytic activity, via both individual and cooperative effects, hence providing insights into the mechanisms through which these two mutations are epistatically coupled.}, }
@article {pmid35352981, year = {2022}, author = {Li, Y and Liu, Y and Singh, J and Tangprasertchai, NS and Trivedi, R and Fang, Y and Qin, PZ}, title = {Site-Specific Labeling Reveals Cas9 Induces Partial Unwinding Without RNA/DNA Pairing in Sequences Distal to the PAM.}, journal = {The CRISPR journal}, volume = {5}, number = {2}, pages = {341-352}, doi = {10.1089/crispr.2021.0100}, pmid = {35352981}, issn = {2573-1602}, mesh = {*CRISPR-Cas Systems/genetics ; DNA/chemistry/genetics ; Endonucleases/genetics ; Gene Editing ; *RNA/chemistry/genetics ; }, abstract = {CRISPR-Cas9 is an RNA-guided nuclease that has been widely adapted for genome engineering. A key determinant in Cas9 target selection is DNA duplex unwinding to form an R-loop, in which the single-stranded RNA guide hybridizes with one of the DNA strands. To advance understanding on DNA unwinding by Cas9, we combined two types of spectroscopic label, 2-aminopurine and nitroxide spin-label, to investigate unwinding at a specific DNA base pair induced by Streptococcus pyogenes Cas9. Data obtained with RNA guide lengths varying from 13 to 20 nucleotide revealed that the DNA segment distal to the protospacer adjacent motif can adopt a "partial unwinding" state, in which a mixture of DNA-paired and DNA-unwound populations exist in equilibrium. Significant unwinding can occur at positions not supported by RNA/DNA pairing, and the degree of unwinding depends on RNA guide length and modulates DNA cleavage activity. The results shed light on Cas9 target selection and may inform developments of genome-engineering strategies.}, }
@article {pmid35351985, year = {2022}, author = {Sridhara, S and Rai, J and Whyms, C and Goswami, H and He, H and Woodside, W and Terns, MP and Li, H}, title = {Structural and biochemical characterization of in vivo assembled Lactococcus lactis CRISPR-Csm complex.}, journal = {Communications biology}, volume = {5}, number = {1}, pages = {279}, pmid = {35351985}, issn = {2399-3642}, support = {S10 RR025080/RR/NCRR NIH HHS/United States ; U24 GM116788/GM/NIGMS NIH HHS/United States ; S10 RR024564/RR/NCRR NIH HHS/United States ; GM099604//U.S. Department of Health &amp; Human Services | NIH | Center for Information Technology (Center for Information Technology, National Institutes of Health)/ ; R01 GM099604/GM/NIGMS NIH HHS/United States ; R35 GM118160/GM/NIGMS NIH HHS/United States ; }, mesh = {Adenosine Triphosphate ; Bacterial Proteins/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Lactococcus lactis/genetics/metabolism ; RNA ; }, abstract = {The small RNA-mediated immunity in bacteria depends on foreign RNA-activated and self RNA-inhibited enzymatic activities. The multi-subunit Type III-A CRISPR-Cas effector complex (Csm) exemplifies this principle and is in addition regulated by cellular metabolites such as divalent metals and ATP. Recognition of the foreign or cognate target RNA (CTR) triggers its single-stranded deoxyribonuclease (DNase) and cyclic oligoadenylate (cOA) synthesis activities. The same activities remain dormant in the presence of the self or non-cognate target RNA (NTR) that differs from CTR only in its 3'-protospacer flanking sequence (3'-PFS). Here we employ electron cryomicroscopy (cryoEM), functional assays, and comparative cross-linking to study in vivo assembled mesophilic Lactococcus lactis Csm (LlCsm) at the three functional states: apo, the CTR- and the NTR-bound. Unlike previously studied Csm complexes, we observed binding of 3'-PFS to Csm in absence of bound ATP and analyzed the structures of the four RNA cleavage sites. Interestingly, comparative crosslinking results indicate a tightening of the Csm3-Csm4 interface as a result of CTR but not NTR binding, reflecting a possible role of protein dynamics change during activation.}, }
@article {pmid35351888, year = {2022}, author = {Jia, K and Cui, YR and Huang, S and Yu, P and Lian, Z and Ma, P and Liu, J}, title = {Phage peptides mediate precision base editing with focused targeting window.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {1662}, pmid = {35351888}, issn = {2041-1723}, support = {31600686//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {Alleles ; Animals ; *Bacteriophages/genetics ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Mice ; Peptides/genetics ; }, abstract = {Base editors (BEs) are genome engineering tools that can generate nucleotide substitutions without introducing double-stranded breaks (DSBs). A variety of strategies have been developed to improve the targeting scope and window of BEs. In a previous study, we found that a bacteriophage-derived peptide, referred to as G8PPD, could improve the specificity of Cas9 nuclease. Herein, we investigate the applicability of G8PPD as molecular modulators of BEs. We show that G8PPD can improve cytidine base editor (CBEs) and adenine base editor (ABE) to more focused targeting windows. Notably, in a cell-based disease model, G8PPD increases the percentage of perfectly edited gene alleles by BEs from less than 4% to more than 38% of the whole population. In addition, G8PPD can improve the targeting scope of BE in mouse embryos. In summary, our study presents the peptidyl modulators that can improve BEs for precision base editing.}, }
@article {pmid35351879, year = {2022}, author = {Li, X and Zhou, L and Gao, BQ and Li, G and Wang, X and Wang, Y and Wei, J and Han, W and Wang, Z and Li, J and Gao, R and Zhu, J and Xu, W and Wu, J and Yang, B and Sun, X and Yang, L and Chen, J}, title = {Highly efficient prime editing by introducing same-sense mutations in pegRNA or stabilizing its structure.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {1669}, pmid = {35351879}, issn = {2041-1723}, support = {2019YFA0802804//Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)/ ; 2018ZX10731-101-001-010//Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)/ ; 2019YFA0802804//Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)/ ; 2018YFA0801401//Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)/ ; 2018YFC1004602//Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)/ ; 32070170//National Natural Science Foundation of China (National Science Foundation of China)/ ; 31925011//National Natural Science Foundation of China (National Science Foundation of China)/ ; 91940306//National Natural Science Foundation of China (National Science Foundation of China)/ ; 31822016//National Natural Science Foundation of China (National Science Foundation of China)/ ; 21JC1404600//Science and Technology Commission of Shanghai Municipality (Shanghai Municipal Science and Technology Commission)/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; INDEL Mutation ; Mutation ; RNA-Directed DNA Polymerase/genetics ; }, abstract = {Prime editor (PE), which is developed by combining Cas9 nickase and an engineered reverse transcriptase, can mediate all twelve types of base substitutions and small insertions or deletions in living cells but its efficiency remains low. Here, we develop spegRNA by introducing same-sense mutations at proper positions in the reverse-transcription template of pegRNA to increase PE's base-editing efficiency up-to 4,976-fold (on-average 353-fold). We also develop apegRNA by altering the pegRNA secondary structure to increase PE's indel-editing efficiency up-to 10.6-fold (on-average 2.77-fold). The spegRNA and apegRNA can be combined to further enhance editing efficiency. When spegRNA and apegRNA are used in PE3 and PE5 systems, the efficiencies of sPE3, aPE3, sPE5 and aPE5 systems are all enhanced significantly. The strategies developed in this study realize highly efficient prime editing at certain previously uneditable sites.}, }
@article {pmid35350901, year = {2022}, author = {Wheatley, MS and Wang, Q and Wei, W and Bottner-Parker, K and Zhao, Y and Yang, Y}, title = {Cas12a-based diagnostics for potato purple top disease complex associated with infection by 'Candidatus Phytoplasma trifolii'-related strains.}, journal = {Plant disease}, volume = {}, number = {}, pages = {}, doi = {10.1094/PDIS-09-21-2119-RE}, pmid = {35350901}, issn = {0191-2917}, abstract = {'Candidatus Phytoplasma trifolii' is a cell wall-less phytopathogenic bacterium that infects many agriculturally important plant species such as alfalfa, clover, eggplant, pepper, potato, and tomato. The phytoplasma is responsible for repeated outbreaks of potato purple top (PPT) and potato witches' broom (PWB) that occurred along the Pacific Coast of the United States since 2002, inflicting significant economic losses. To effectively manage these phytoplasmal diseases, it is important to develop diagnostic tools for specific, sensitive and rapid detection of the pathogens. Here we report the development of a DNA endonuclease targeted CRISPR trans reporter (DETECTR) assay that couples isothermal amplification and Cas12a trans-cleavage of fluorescent oligonucleotide reporter for highly sensitive and specific detection of 'Candidatus Phytoplasma trifolii'-related strains responsible for PPT and PWB. The DETECTR assay was capable of specifically detecting the 16S-23S ribosomal DNA (rDNA) intergenic transcribed spacer (ITS) sequences from PPT- and PWB-diseased samples at the attomolar sensitivity level. Furthermore, the DETECTR strategy allows flexibility to capture assay outputs with fluorescent microplate reader or lateral flow assay for potentially high-throughput and/or field-deployable disease diagnostics.}, }
@article {pmid35349718, year = {2022}, author = {Konstantakos, V and Nentidis, A and Krithara, A and Paliouras, G}, title = {CRISPR-Cas9 gRNA efficiency prediction: an overview of predictive tools and the role of deep learning.}, journal = {Nucleic acids research}, volume = {50}, number = {7}, pages = {3616-3637}, pmid = {35349718}, issn = {1362-4962}, mesh = {CRISPR-Cas Systems ; *Deep Learning ; *Gene Editing/methods ; *RNA, Guide/genetics ; }, abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has become a successful and promising technology for gene-editing. To facilitate its effective application, various computational tools have been developed. These tools can assist researchers in the guide RNA (gRNA) design process by predicting cleavage efficiency and specificity and excluding undesirable targets. However, while many tools are available, assessment of their application scenarios and performance benchmarks are limited. Moreover, new deep learning tools have been explored lately for gRNA efficiency prediction, but have not been systematically evaluated. Here, we discuss the approaches that pertain to the on-target activity problem, focusing mainly on the features and computational methods they utilize. Furthermore, we evaluate these tools on independent datasets and give some suggestions for their usage. We conclude with some challenges and perspectives about future directions for CRISPR-Cas9 guide design.}, }
@article {pmid35349689, year = {2022}, author = {Zhao, D and Jiang, G and Li, J and Chen, X and Li, S and Wang, J and Zhou, Z and Pu, S and Dai, Z and Ma, Y and Bi, C and Zhang, X}, title = {Imperfect guide-RNA (igRNA) enables CRISPR single-base editing with ABE and CBE.}, journal = {Nucleic acids research}, volume = {50}, number = {7}, pages = {4161-4170}, pmid = {35349689}, issn = {1362-4962}, mesh = {Adenine/metabolism ; Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing/methods ; *RNA, Guide/genetics ; }, abstract = {CRISPR base editing techniques tend to edit multiple bases in the targeted region, which is a limitation for precisely reverting disease-associated single-nucleotide polymorphisms (SNPs). We designed an imperfect gRNA (igRNA) editing methodology, which utilized a gRNA with one or more bases that were not complementary to the target locus to direct base editing toward the generation of a single-base edited product. Base editing experiments illustrated that igRNA editing with CBEs greatly increased the single-base editing fraction relative to normal gRNA editing with increased editing efficiencies. Similar results were obtained with an adenine base editor (ABE). At loci such as DNMT3B, NSD1, PSMB2, VIATA hs267 and ANO5, near-perfect single-base editing was achieved. Normally an igRNA with good single-base editing efficiency could be selected from a set of a few igRNAs, with a simple protocol. As a proof-of-concept, igRNAs were used in the research to construct cell lines of disease-associated SNP causing primary hyperoxaluria construction research. This work provides a simple strategy to achieve single-base base editing with both ABEs and CBEs and overcomes a key obstacle that limits the use of base editors in treating SNP-associated diseases or creating disease-associated SNP-harboring cell lines and animal models.}, }
@article {pmid35349073, year = {2022}, author = {Das, S and Bombaywala, S and Srivastava, S and Kapley, A and Dhodapkar, R and Dafale, NA}, title = {Genome plasticity as a paradigm of antibiotic resistance spread in ESKAPE pathogens.}, journal = {Environmental science and pollution research international}, volume = {}, number = {}, pages = {}, pmid = {35349073}, issn = {1614-7499}, abstract = {The major reason behind the spread of antibiotic resistance genes (ARGs) is persistent selective pressure in the environment encountered by bacteria. Genome plasticity plays a crucial role in dissemination of antibiotic resistance among bacterial pathogens. Mobile genetic elements harboring ARGs are reported to dodge bacterial immune system and mediate horizontal gene transfer (HGT) under selective pressure. Residual antibiotic pollutants develop selective pressures that force the bacteria to lose their defense mechanisms (CRISPR-cas) and acquire resistance. The present study targets the ESKAPE organisms (namely, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) causing various nosocomial infections and emerging multidrug-resistant species. The role of CRISPR-cas systems in inhibition of HGT in prokaryotes and its loss due to presence of various stressors in the environment is also focused in the study. IncF and IncH plasmids were identified in all strains of E. faecalis and K. pneumoniae, carrying Beta-lactam and fluoroquinolone resistance genes, whereas sal3, phiCTX, and SEN34 prophages harbored aminoglycoside resistance genes (aadA, aac). Various MGEs present in selected environmental niches that aid the bacterial genome plasticity and transfer of ARGs contributing to its spread are also identified.}, }
@article {pmid35348649, year = {2022}, author = {Dong, C and Wang, X and Ma, C and Zeng, Z and Pu, DK and Liu, S and Wu, CS and Chen, S and Deng, Z and Guo, FB}, title = {Anti-CRISPRdb v2.2: an online repository of anti-CRISPR proteins including information on inhibitory mechanisms, activities and neighbors of curated anti-CRISPR proteins.}, journal = {Database : the journal of biological databases and curation}, volume = {2022}, number = {}, pages = {}, doi = {10.1093/database/baac010}, pmid = {35348649}, issn = {1758-0463}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Viral Proteins/genetics ; }, abstract = {We previously released the Anti-CRISPRdb database hosting anti-CRISPR proteins (Acrs) and associated information. Since then, the number of known Acr families, types, structures and inhibitory activities has accumulated over time, and Acr neighbors can be used as a candidate pool for screening Acrs in further studies. Therefore, we here updated the database to include the new available information. Our newly updated database shows several improvements: (i) it comprises more entries and families because it includes both Acrs reported in the most recent literatures and Acrs obtained via performing homologous alignment; (ii) the prediction of Acr neighbors is integrated into Anti-CRISPRdb v2.2, and users can identify novel Acrs from these candidates; and (iii) this version includes experimental information on the inhibitory strength and stage for Acr-Cas/Acr-CRISPR pairs, motivating the development of tools for predicting specific inhibitory abilities. Additionally, a parameter, the rank of codon usage bias (CUBRank), was proposed and provided in the new version, which showed a positive relationship with predicted result from AcRanker; hence, it can be used as an indicator for proteins to be Acrs. CUBRank can be used to estimate the possibility of genes occurring within genome island-a hotspot hosting potential genes encoding Acrs. Based on CUBRank and Anti-CRISPRdb, we also gave the first glimpse for the emergence of Acr genes (acrs).<br><br>DATABASE URL: http://guolab.whu.edu.cn/anti-CRISPRdb.}, }
@article {pmid35347685, year = {2022}, author = {Frey, N and Schwank, G}, title = {CRISPR-Based Screening in Three-Dimensional Organoid Cultures to Identify TGF-β Pathway Regulators.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2488}, number = {}, pages = {99-111}, pmid = {35347685}, issn = {1940-6029}, mesh = {Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Intestines ; *Organoids ; *Transforming Growth Factor beta/metabolism ; Wnt Signaling Pathway/genetics ; }, abstract = {The CRISPR/Cas technology has revolutionized forward genetic screening, and thereby facilitated genetic dissection of cellular processes and pathways. TGF-β signaling is a highly conserved cascade involved in development, regeneration, and diseases such as cancer. Even though many core components of the signaling cascade have already been described, several context-dependent pathway modulators remain unknown. To address this knowledge gap, we have recently developed a CRISPR screening approach for identifying TGF-β pathway regulators in three-dimensional organoid culture systems. Here, we provide a detailed protocol describing this approach in human intestinal organoids. With adaptations, this screening method could also be applied to other organoid types, and to other signaling cascades such as EGF or WNT signaling, thereby uncovering important mechanism in regeneration and disease.}, }
@article {pmid35347684, year = {2022}, author = {Huang, Z and Loewer, A}, title = {Generating Somatic Knockout Cell Lines with CRISPR-Cas9 Technology to Investigate SMAD Signaling.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2488}, number = {}, pages = {81-97}, pmid = {35347684}, issn = {1940-6029}, mesh = {*CRISPR-Cas Systems/genetics ; Cell Line ; Humans ; *Signal Transduction/genetics ; Technology ; }, abstract = {Genome engineering provides a powerful tool to explore TGF-β/SMAD signaling by enabling the deletion and modification of critical components of the pathway. Over the past years, CRISPR-Cas9 technology has matured and can now be used to routinely generate knockout cell lines. Here, we describe a method to design and generate deletions of genes from the SMAD pathway in somatic human cell lines based on homologous recombination.}, }
@article {pmid35344733, year = {2022}, author = {Rusni, S and Sassa, M and Takagi, T and Kinoshita, M and Takehana, Y and Inoue, K}, title = {Establishment of cytochrome P450 1a gene-knockout Javanese medaka, Oryzias javanicus, which distinguishes toxicity modes of the polycyclic aromatic hydrocarbons, pyrene and phenanthrene.}, journal = {Marine pollution bulletin}, volume = {178}, number = {}, pages = {113578}, doi = {10.1016/j.marpolbul.2022.113578}, pmid = {35344733}, issn = {1879-3363}, mesh = {Animals ; Cytochrome P-450 CYP1A1/genetics/metabolism ; Cytochrome P-450 Enzyme System/metabolism ; Indonesia ; *Oryzias/genetics ; *Phenanthrenes/metabolism/toxicity ; *Polycyclic Aromatic Hydrocarbons/analysis ; Pyrenes/metabolism/toxicity ; *Water Pollutants, Chemical/analysis ; }, abstract = {Cytochrome P450 1a (Cyp1a) is an important enzyme for metabolism of organic pollutants. To understand its reaction to polycyclic aromatic hydrocarbons (PAHs), we knocked out this gene in a marine model fish, Javanese medaka, Oryzias javanicus, using the CRISPR/Cas 9 system. A homozygous mutant (KO) strain with a four-base deletion was established using an environmental DNA (eDNA)-based genotyping technique. Subsequently, KO, heterozygous mutant (HT), and wild-type (WT) fish were exposed to model pollutants, pyrene and phenanthrene, and survivorship and swimming behavior were analyzed. Compared to WT, KO fish were more sensitive to pyrene, suggesting that Cyp1a transforms pyrene into less toxic metabolites. Conversely, WT fish were sensitive to phenanthrene, suggesting that metabolites transformed by Cyp1a are more toxic than the original compound. HT fish showed intermediate results. Thus, comparative use of KO and WT fish can distinguish modes of pollutant toxicity, providing a deeper understanding of fish catabolism of environmental pollutants.}, }
@article {pmid35343817, year = {2022}, author = {Brackett, NF and Davis, BW and Adli, M and Pomés, A and Chapman, MD}, title = {Evolutionary Biology and Gene Editing of Cat Allergen, Fel d 1.}, journal = {The CRISPR journal}, volume = {5}, number = {2}, pages = {213-223}, doi = {10.1089/crispr.2021.0101}, pmid = {35343817}, issn = {2573-1602}, mesh = {*Allergens/chemistry/genetics ; Animals ; Biology ; CRISPR-Cas Systems/genetics ; Cats ; Gene Editing ; Glycoproteins/chemistry/genetics ; *Hypersensitivity/genetics/therapy ; }, abstract = {Allergy to domestic cat affects up to 15% of the population, and sensitization to cat allergen is associated with asthma. Despite the pervasiveness of cat allergic disease, current treatments have limited impact. Here, we present a bioinformatics analysis of the major cat allergen, Fel d 1, and demonstrate proof of principle for CRISPR gene editing of the allergen. Sequence and structural analyses of Fel d 1 from 50 domestic cats identified conserved coding regions in genes CH1 and CH2 suitable for CRISPR editing. Comparative analyses of Fel d 1 and orthologous sequences from eight exotic felid species determined relatively low-sequence identities for CH1 and CH2, and implied that the allergen may be nonessential for cats, given the apparent lack of evolutionary conservation. In vitro knockouts of domestic cat Fel d 1 using CRISPR-Cas9 yielded editing efficiencies of up to 55% and found no evidence of editing at predicted potential off-target sites. Taken together, our data indicate that Fel d 1 is both a rational and viable candidate for gene deletion, which may profoundly benefit cat allergy sufferers by removing the major allergen at the source.}, }
@article {pmid35343224, year = {2022}, author = {Tian, H and Niu, H and Luo, J and Yao, W and Chen, X and Wu, J and Geng, Y and Gao, W and Lei, A and Gao, Z and Tian, X and Zhao, X and Shi, H and Li, C and Hua, J}, title = {Knockout of Stearoyl-CoA Desaturase 1 Decreased Milk Fat and Unsaturated Fatty Acid Contents of the Goat Model Generated by CRISPR/Cas9.}, journal = {Journal of agricultural and food chemistry}, volume = {70}, number = {13}, pages = {4030-4043}, doi = {10.1021/acs.jafc.2c00642}, pmid = {35343224}, issn = {1520-5118}, mesh = {Animals ; CRISPR-Cas Systems ; Fatty Acids/metabolism ; Fatty Acids, Unsaturated/metabolism ; *Goats/metabolism ; *Milk/chemistry ; *Stearoyl-CoA Desaturase/genetics/metabolism ; }, abstract = {Goat milk contains a rich source of nutrients, especially unsaturated fatty acids. However, the regulatory mechanism of milk fat and fatty acid synthesis remains unclear. Stearoyl-CoA desaturase 1 (SCD1) is the key enzyme catalyzing monounsaturated fatty acid synthesis and is essential for milk lipid metabolism. To explore milk lipid synthesis mechanism in vivo, SCD1-knockout goats were generated through CRISPR/Cas9 technology for the first time. SCD1 deficiency did not influence goat growth or serum biochemistry. Plasma phosphatidylcholines increased by lipidomics after SCD1 knockout in goats. Whole-blood RNA-seq indicated alterations in biosynthesis of unsaturated fatty acid synthesis, cAMP, ATPase activity, and Wnt signaling pathways. In SCD1-knockout goats, milk fat percentage and unsaturated fatty acid levels were reduced but other milk components were unchanged. Milk lipidomics revealed decreased triacylglycerols and diacylglycerols levels, and the differential abundance of lipids were enriched in glycerolipid, glycerophospholipids, and thermogenesis metabolism pathways. In milk fat globules, the expression levels of genes related to fatty acid and TAG synthesis including SREBP1 were reduced. ATP content and AMPK activity were promoted, and p-p70S6K protein level was suppressed in SCD1-knockout goat mammary epithelial cells, suggesting that SCD1 affected milk lipid metabolism by influencing AMPK-mTORC1/p70S6K-SREBP1 pathway. The integrative analysis of gene expression levels and lipidomics of milk revealed a crucial role of SCD1 in glycerolipids and glycerophospholipids metabolism pathways. Our observations indicated that SCD1 regulated the synthesis of milk fat and unsaturated fatty acid in goat by affecting lipid metabolism gene expression and lipid metabolic pathways. These findings would be essential for improving goat milk nutritional value which is beneficial to human health.}, }
@article {pmid35343100, year = {2022}, author = {Zheng, F and Chen, Z and Li, J and Wu, R and Zhang, B and Nie, G and Xie, Z and Zhang, H}, title = {A Highly Sensitive CRISPR-Empowered Surface Plasmon Resonance Sensor for Diagnosis of Inherited Diseases with Femtomolar-Level Real-Time Quantification.}, journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)}, volume = {9}, number = {14}, pages = {e2105231}, doi = {10.1002/advs.202105231}, pmid = {35343100}, issn = {2198-3844}, support = {JCYJ20180508152903208//The Science and Technology Project of Shenzhen/ ; 2019B1515120043//Guangdong Basic and Applied Basic Research Foundation/ ; 2020A151501612//Natural Science Foundation of Guangdong Province/ ; JCYJ201904//Longhua District Science and Innovation Commission Project Grants of Shenzhen/ ; KCXFZ20201221173413038//Science and Technology Innovation Commission of Shenzhen/ ; }, mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Humans ; *Nucleic Acids ; RNA, Guide/genetics/metabolism ; Surface Plasmon Resonance ; }, abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) molecular system has emerged as a promising technology for the detection of nucleic acids. Herein, the development of a surface plasmon resonance (SPR) sensor that is functionalized with a layer of locally grown graphdiyne film, achieving excellent sensing performance when coupled with catalytically deactivated CRISPR-associated protein 9 (dCas9), is reported. dCas9 protein is immobilized on the sensor surface and complexed with a specific single-guide RNA, enabling the amplification-free detection of target sequences within genomic DNA. The sensor, termed CRISPR-SPR-Chip, is used to successfully analyze recombinant plasmids with only three-base mutations with a limit of detection as low as 1.3 fM. Real-time monitoring CRISPR-SPR-Chip is used to analyze clinical samples of patients with Duchenne muscular dystrophy with two exon deletions, which are detected without any pre-amplification step, yielding significantly positive results within 5 min. The ability of this novel CRISPR-empowered SPR (CRISPR-eSPR) sensing platform to rapidly, precisely, sensitively, and specifically detect a target gene sequence provides a new on-chip optic approach for clinical gene analysis.}, }
@article {pmid35341983, year = {2022}, author = {Li, C and Chu, W and Gill, RA and Sang, S and Shi, Y and Hu, X and Yang, Y and Zaman, QU and Zhang, B}, title = {Computational tools and resources for CRISPR/Cas genome editing.}, journal = {Genomics, proteomics &amp; bioinformatics}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.gpb.2022.02.006}, pmid = {35341983}, issn = {2210-3244}, abstract = {The past decade has witnessed a rapid evolution in identifying more versatile clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) nucleases and their functional variants as well as in developing precise CRISPR/Cas-derived genome editors. The programmable and robust features of the genomic editors provide an effective RNA-guided platform for fundamental life science research and subsequent applications in diverse scenarios, including biomedical innovation and targeted crop improvement. One of the most essential principles is to guide alterations in genomic sequences or genes in the intended manner without undesired off-target impacts, which strongly depends on the efficiency and specificity of single guide RNA (sgRNA)-directed recognition of targeted DNA sequences. Recent advances in empirical scoring algorithms and machine learning models have facilitated sgRNA design and off-target prediction. In this review, we first briefly introduced the different features of CRISPR/Cas tools that should be taken into consideration to achieve specific purposes. Secondly, we focused on the computer-assisted tools and resources that are widely used in designing sgRNAs and analyzing CRISPR/Cas-induced on- and off-target mutations. Thirdly, we provide insights on the limitations of available computational tools that surely help researchers of this field for further optimization. Lastly, we suggested a simple but effective workflow for choosing and applying web-based resources and tools for CRISPR/Cas genome editing.}, }
@article {pmid35341958, year = {2022}, author = {Wang, S and Hu, J and Sui, C and He, G and Qu, Z and Chen, X and Wang, Y and Guo, D and Liu, X}, title = {Accuracy of clustered regularly interspaced short palindromic repeats (CRISPR) to diagnose COVID-19, a meta-analysis.}, journal = {Microbial pathogenesis}, volume = {165}, number = {}, pages = {105498}, pmid = {35341958}, issn = {1096-1208}, mesh = {*COVID-19/diagnosis ; CRISPR-Cas Systems ; Humans ; }, abstract = {OBJECTIVE: To estimate the accuracy of clustered regularly interspaced short palindromic repeats (CRISPR) in determining coronavirus disease-19 (COVID-19).<br><br>METHODS: As of January 31, 2022, PubMed, Web of Science, Embase, Science Direct, Wiley and Springer Link were searched. Sensitivity, specificity, likelihood ratio (LR), diagnostic odds ratio (DOR) and area under the summary receiver-operating characteristic (AUC) curve were used to assess the accuracy of CRISPR.<br><br>RESULTS: According to the inclusion criteria, 5857 patients from 54 studies were included in this meta-analysis. The pooled sensitivity, specificity and AUC were 0.98, 1.00 and 1.00, respectively. For CRISPR-associated (Cas) proteins-12, the sensitivity, specificity was 0.96, 1.00, respectively. For Cas-13, the sensitivity and specificity were 0.99 and 0.99.<br><br>CONCLUSION: This meta-analysis showed that the diagnostic performance of CRISPR is close to the gold standard, and it is expected to meet the Point of care requirements in resource poor areas.}, }
@article {pmid35340673, year = {2022}, author = {Alduhaidhawi, AHM and AlHuchaimi, SN and Al-Mayah, TA and Al-Ouqaili, MTS and Alkafaas, SS and Muthupandian, S and Saki, M}, title = {Prevalence of CRISPR-Cas Systems and Their Possible Association with Antibiotic Resistance in Enterococcus faecalis and Enterococcus faecium Collected from Hospital Wastewater.}, journal = {Infection and drug resistance}, volume = {15}, number = {}, pages = {1143-1154}, pmid = {35340673}, issn = {1178-6973}, abstract = {Purpose: This study aimed to evaluate the presence of CRISPR-Cas system genes and their possible association with antibiotic resistance patterns of Enterococcus faecalis and Enterococcus faecium species isolated from hospital wastewater (HWW) samples of several hospitals.<br><br>Methods: HWW samples (200 mL) were collected from wastewater discharged from different hospitals from October 2020 to March 2021. The isolation and identification of enterococci species were performed by standard bacteriology tests and polymerase chain reaction (PCR). Antibiotic resistance was determined using the disc diffusion. The presence of various CRISPR-Cas systems was investigated by PCR. The association of the occurrence of CRISPR-Cas systems with antibiotic resistance was analyzed with appropriate statistical tests.<br><br>Results: In total, 85 different enterococci species were isolated and identified using phenotypic methods. The results of PCR confirmed the prevalence of 50 (58.8%) E. faecalis and 35 (41.2%) E. faecium, respectively. In total, 54 (63.5%) of 85 isolates showed the presence of CRISPR-Cas loci. The incidence of CRISPR-Cas was more common in E. faecalis. CRISPR1, CRISPR2, and CRISPR3 were present in 35 (41.2%), 47 (55.3%), and 30 (35.3%) enterococci isolates, respectively. The CRISPR-Cas positive isolates showed significant lower resistance rates against vancomycin, ampicillin, chloramphenicol, erythromycin, rifampin, teicoplanin, tetracycline, imipenem, tigecycline, and trimethoprim-sulfamethoxazole in comparison with CRISPR-Cas negative isolates. The results showed that the presence of CRISPR-Cas genes was lower in multidrug-resistant (MDR) isolates (53.1%, n = 26/49) compared to the non-MDR enterococci isolates (77.8%, n = 28/36) (P = 0.023).<br><br>Conclusion: This study revealed the higher prevalence of E. faecalis than E. faecium in HWWs. Also, the lack of CRISPR-Cas genes was associated with more antibiotic resistance rates and multidrug resistance in E. faecalis and E. faecium isolates with HWW origin.}, }
@article {pmid35339825, year = {2022}, author = {Geng, B and Wang, X and Park, KH and Lee, KE and Kim, J and Chen, P and Zhou, X and Tan, T and Yang, C and Zou, X and Janssen, PM and Cao, L and Ye, L and Wang, X and Cai, C and Zhu, H}, title = {UCHL1 protects against ischemic heart injury via activating HIF-1α signal pathway.}, journal = {Redox biology}, volume = {52}, number = {}, pages = {102295}, doi = {10.1016/j.redox.2022.102295}, pmid = {35339825}, issn = {2213-2317}, mesh = {Animals ; Escherichia coli ; *Heart Injuries ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics ; *Induced Pluripotent Stem Cells ; Mice ; *Myocardial Infarction/genetics/pathology ; Signal Transduction ; Ubiquitin Thiolesterase/genetics ; }, abstract = {Ubiquitin carboxyl-terminal esterase L1 (UCHL1) has been thought to be a neuron specific protein and shown to play critical roles in Parkinson's Disease and stroke via de-ubiquiting and stabilizing key pathological proteins, such as α-synuclein. In the present study, we found that UCHL1 was significantly increased in both mouse and human cardiomyocytes following myocardial infarction (MI). When LDN-57444, a pharmacological inhibitor of UCHL1, was used to treat mice subjected to MI surgery, we found that administration of LDN-57444 compromised cardiac function when compared with vehicle treated hearts, suggesting a potential protective role of UCHL1 in response to MI. When UCHL1 was knockout by CRISPR/Cas 9 gene editing technique in human induced pluripotent stem cells (hiPSCs), we found that cardiomyocytes derived from UCHL1-/- hiPSCs were more susceptible to hypoxia/re-oxygenation induced injury as compared to wild type cardiomyocytes. To study the potential targets of UCHL1, a BioID based proximity labeling approach followed by mass spectrum analysis was performed. The result suggested that UCHL1 could bind to and stabilize HIF-1α following MI. Indeed, expression of HIF-1α was lower in UCHL1-/- cells as determined by Western blotting and HIF-1α target genes were also suppressed in UCHL1-/- cells as quantified by real time RT-PCR. Recombinant UCHL1 (rUCHL1) protein was purified by E. Coli fermentation and intraperitoneally (I.P.) delivered to mice. We found that administration of rUCHL1 could significantly preserve cardiac function following MI as compared to control group. Finally, adeno associated virus mediated cardiac specific UCHL1 delivery (AAV9-cTNT-m-UCHL1) was performed in neonatal mice. UCHL1 overexpressing hearts were more resistant to MI injury as compare to the hearts infected with control virus. In summary, our data revealed a novel protective role of UCHL1 on MI via stabilizing HIF-1α and promoting HIF-1α signaling.}, }
@article {pmid35339823, year = {2022}, author = {Liu, C and Ren, L and Li, X and Fan, N and Chen, J and Zhang, D and Yang, W and Ding, S and Xu, W and Min, X}, title = {Self-electrochemiluminescence biosensor based on CRISPR/Cas12a and PdCuBP@luminol nanoemitter for highly sensitive detection of cytochrome c oxidase subunit III gene of acute kidney injury.}, journal = {Biosensors &amp; bioelectronics}, volume = {207}, number = {}, pages = {114207}, doi = {10.1016/j.bios.2022.114207}, pmid = {35339823}, issn = {1873-4235}, mesh = {*Acute Kidney Injury ; *Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; Electrochemical Techniques/methods ; Electron Transport Complex IV ; Female ; Humans ; Limit of Detection ; Luminescent Measurements/methods ; Luminol ; Male ; }, abstract = {The cytochrome c oxidase subunit III (COX III) gene is a powerful biomarker for the early diagnosis of acute kidney injury. However, current methods for COX III gene detection are usually laborious and time-consuming, with limited sensitivity. Herein, we report a novel self-electrochemiluminescence (ECL) biosensor for highly sensitive detection of the COX III gene based on CRISPR/Cas12a and nanoemitters of luminol-loaded multicomponent metal-metalloid PdCuBP alloy mesoporous nanoclusters. The nanoemitter with excellent self-ECL in neutral media exhibited a high specific surface area for binding luminol and outstanding oxidase-like catalytic activity toward dissolved O2. Meanwhile, the CRISPR/Cas12a system, as a target-trigger, was employed to specifically recognize the COX III gene and efficiently cleave the interfacial quencher of dopamine-labeled hairpin DNA. As a result, the ECL biosensor showed superior analytical performance for COX III gene detection without exogenous coreactant. Benefiting from the high-efficiency ECL emission of the nanoemitter and Cas12a-mediated interfacial cleavage of the quencher, the developed ECL biosensor exhibited high sensitivity to COX III with a low detection limit of 0.18 pM. The established ECL biosensing method possessed excellent practical performance in urine samples. Meaningfully, the proposed strategy presents promising prospects for nucleic acid detection in the field of clinical diagnostics.}, }
@article {pmid35339702, year = {2022}, author = {Clemmensen, SE and Kromphardt, KJK and Frandsen, RJN}, title = {Marker-free CRISPR-Cas9 based genetic engineering of the phytopathogenic fungus, Penicillium expansum.}, journal = {Fungal genetics and biology : FG &amp; B}, volume = {160}, number = {}, pages = {103689}, doi = {10.1016/j.fgb.2022.103689}, pmid = {35339702}, issn = {1096-0937}, mesh = {CRISPR-Cas Systems ; Genetic Engineering ; *Malus ; *Penicillium/genetics/metabolism ; }, abstract = {Filamentous fungi are prolific producers of secondary metabolites (SecMets), including compounds with antibiotic properties, like penicillin, that allows the producing fungus to combat competitors in a shared niche. However, the biological function of the majority of these small complex metabolites for the producing fungi remains unclear (Macheleidt et al., 2016). In an effort to address this lack of knowledge, we have chosen to study the microbial community of moldy apples in the hope of shedding more light on the role of SecMets for the dynamics of the microbial community. Penicillium expansum is one of the prevalent fungal species in this system, and in co-culture experiments with other apple fungal pathogens, we have observed up- and downregulation of several SecMets when compared to monocultures. However, molecular genetic dissection of the observed changes is challenging, and new methodologies for targeted genetic engineering in P. expansum are needed. In the current study, we have established a CRISPR-Cas9 dependent genetic engineering toolbox for the targeted genetic manipulation of P. expansum to allow for single-step construction of marker-free strains. The method and effect of different combinations of a Cas9-sgRNA expressing plasmids and repair template substrates in the NHEJ-proficient WT strain is tested by targeted deletion of melA, encoding a PKS responsible for pigment formation, which upon deletion resulted in white mutants. Co-transformation with a linear double-stranded DNA fragment consisting of two 2 kb homology arms flanking the PKS gene proved to be the most efficient strategy with 100% confirmed deletions by diagnostic PCR. Shorter homology arms (500-1000 bp) resulted in 20-30% deletion efficiency. Furthermore, we demonstrate the application of the CRISPR-Cas9 method for targeted deletion of biosynthetic genes without a visible phenotype, insertion of a visual reporter-encoding gene (mRFP), and overexpression of biosynthetic genes. Combined, these tools will advance in enabling the deciphering of SecMet biosynthetic pathways, provide in situ insight into when and where SecMets are produced, and provide an avenue to study the role of P. expansum SecMets in shaping the microbial community development on moldy apples via marker-free targeted genetic engineering of P. expansum.}, }
@article {pmid35338974, year = {2022}, author = {Fapohunda, FO and Qiao, S and Pan, Y and Wang, H and Liu, Y and Chen, Q and Lü, P}, title = {CRISPR Cas system: A strategic approach in detection of nucleic acids.}, journal = {Microbiological research}, volume = {259}, number = {}, pages = {127000}, doi = {10.1016/j.micres.2022.127000}, pmid = {35338974}, issn = {1618-0623}, mesh = {*CRISPR-Cas Systems ; Humans ; *Nucleic Acids ; }, abstract = {Over the decades, rapid nucleic acid detection has been difficult for scientists, especially in microbiology, biotechnology and immunology. Most technologies are finite in sensitivity, specificity or both. Early diagnosis of disease allows swift response, disease monitoring and control of the rapid spread of the disease. However, limited access to test kits, specialized laboratory equipment, and the need for highly skilled personnel has led to a detection downshift. CRISPR-based diagnostic techniques, based on clustered regularly interspaced short palindromic repeats (CRISPR), have recently altered molecular diagnosis. This modern technology is combined or paired with other methods like SHERLOCK, DETECTR, HUDSON, CDetection and so on to detect viruses, bacteria infection and other pathogenic agents, due to its precision and versatility, it can also detect diseases directly from patient samples. Rapid, sensitive, accurate and advanced molecular techniques are much needed since they soothe researchers in diagnosis and detection, and can also be employed in therapeutic treatments. In this review, detailed CRISPR Cas variants were discussed, emphasizing the use of CRISPR system as a tool in diagnostics and nucleic acids detection and the benefits of this robust tool over other amplification methods.}, }
@article {pmid35338236, year = {2022}, author = {Stukenberg, D and Hoff, J and Faber, A and Becker, A}, title = {NT-CRISPR, combining natural transformation and CRISPR-Cas9 counterselection for markerless and scarless genome editing in Vibrio natriegens.}, journal = {Communications biology}, volume = {5}, number = {1}, pages = {265}, pmid = {35338236}, issn = {2399-3642}, support = {BioRoboost project (H2020-NMBP-TR-IND-2018-2020/BIOTEC-01-2018 (CSA), Project ID 210491758)//European Commission (EC)/ ; }, mesh = {CRISPR-Cas Systems ; DNA/genetics ; *Gene Editing/methods ; *Vibrio/genetics ; }, abstract = {The fast-growing bacterium Vibrio natriegens has recently gained increasing attention as a novel chassis organism for fundamental research and biotechnology. To fully harness the potential of this bacterium, highly efficient genome editing methods are indispensable to create strains tailored for specific applications. V. natriegens is able to take up free DNA and incorporate it into its genome by homologous recombination. This highly efficient natural transformation is able to mediate uptake of multiple DNA fragments, thereby allowing for multiple simultaneous edits. Here, we describe NT-CRISPR, a combination of natural transformation with CRISPR-Cas9 counterselection. In two temporally distinct steps, we first performed a genome edit by natural transformation and second, induced CRISPR-Cas9 targeting the wild type sequence, and thus leading to death of non-edited cells. Through cell killing with efficiencies of up to 99.999%, integration of antibiotic resistance markers became dispensable, enabling scarless and markerless edits with single-base precision. We used NT-CRISPR for deletions, integrations and single-base modifications with editing efficiencies of up to 100%. Further, we confirmed its applicability for simultaneous deletion of multiple chromosomal regions. Lastly, we showed that the near PAM-less Cas9 variant SpG Cas9 is compatible with NT-CRISPR and thereby broadens the target spectrum.}, }
@article {pmid35338140, year = {2022}, author = {Krysler, AR and Cromwell, CR and Tu, T and Jovel, J and Hubbard, BP}, title = {Guide RNAs containing universal bases enable Cas9/Cas12a recognition of polymorphic sequences.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {1617}, pmid = {35338140}, issn = {2041-1723}, support = {CIHR-PS-408552//CIHR/Canada ; }, mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; }, abstract = {CRISPR/Cas complexes enable precise gene editing in a wide variety of organisms. While the rigid identification of DNA sequences by these systems minimizes the potential for off-target effects, it consequently poses a problem for the recognition of sequences containing naturally occurring polymorphisms. The presence of genetic variance such as single nucleotide polymorphisms (SNPs) in a gene sequence can compromise the on-target activity of CRISPR systems. Thus, when attempting to target multiple variants of a human gene, or evolved variants of a pathogen gene using a single guide RNA, more flexibility is desirable. Here, we demonstrate that Cas9 can tolerate the inclusion of universal bases in individual guide RNAs, enabling simultaneous targeting of polymorphic sequences. Crucially, we find that specificity is selectively degenerate at the site of universal base incorporation, and remains otherwise preserved. We demonstrate the applicability of this technology to targeting multiple naturally occurring human SNPs with individual guide RNAs and to the design of Cas12a/Cpf1-based DETECTR probes capable of identifying multiple evolved variants of the HIV protease gene. Our findings extend the targeting capabilities of CRISPR/Cas systems beyond their canonical spacer sequences and highlight a use of natural and synthetic universal bases.}, }
@article {pmid35337340, year = {2022}, author = {Das, S and Bano, S and Kapse, P and Kundu, GC}, title = {CRISPR based therapeutics: a new paradigm in cancer precision medicine.}, journal = {Molecular cancer}, volume = {21}, number = {1}, pages = {85}, pmid = {35337340}, issn = {1476-4598}, mesh = {CRISPR-Cas Systems ; Gene Editing ; Humans ; *Neoplasms/genetics/therapy ; *Precision Medicine ; Transcription Activator-Like Effector Nucleases/genetics ; }, abstract = {BACKGROUND: Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) systems are the latest addition to the plethora of gene-editing tools. These systems have been repurposed from their natural counterparts by means of both guide RNA and Cas nuclease engineering. These RNA-guided systems offer greater programmability and multiplexing capacity than previous generation gene editing tools based on zinc finger nucleases and transcription activator like effector nucleases. CRISPR-Cas systems show great promise for individualization of cancer precision medicine.<br><br>MAIN BODY: The biology of Cas nucleases and dead Cas based systems relevant for in vivo gene therapy applications has been discussed. The CRISPR knockout, CRISPR activation and CRISPR interference based genetic screens which offer opportunity to assess functions of thousands of genes in massively parallel assays have been also highlighted. Single and combinatorial gene knockout screens lead to identification of drug targets and synthetic lethal genetic interactions across different cancer phenotypes. There are different viral and non-viral (nanoformulation based) modalities that can carry CRISPR-Cas components to different target organs in vivo.<br><br>CONCLUSION: The latest developments in the field in terms of optimization of performance of the CRISPR-Cas elements should fuel greater application of the latter in the realm of precision medicine. Lastly, how the already available knowledge can help in furtherance of use of CRISPR based tools in personalized medicine has been discussed.}, }
@article {pmid35337108, year = {2022}, author = {Park, HM and Park, Y and Vankerschaver, J and Van Messem, A and De Neve, W and Shim, H}, title = {Rethinking Protein Drug Design with Highly Accurate Structure Prediction of Anti-CRISPR Proteins.}, journal = {Pharmaceuticals (Basel, Switzerland)}, volume = {15}, number = {3}, pages = {}, pmid = {35337108}, issn = {1424-8247}, abstract = {Protein therapeutics play an important role in controlling the functions and activities of disease-causing proteins in modern medicine. Despite protein therapeutics having several advantages over traditional small-molecule therapeutics, further development has been hindered by drug complexity and delivery issues. However, recent progress in deep learning-based protein structure prediction approaches, such as AlphaFold2, opens new opportunities to exploit the complexity of these macro-biomolecules for highly specialised design to inhibit, regulate or even manipulate specific disease-causing proteins. Anti-CRISPR proteins are small proteins from bacteriophages that counter-defend against the prokaryotic adaptive immunity of CRISPR-Cas systems. They are unique examples of natural protein therapeutics that have been optimized by the host-parasite evolutionary arms race to inhibit a wide variety of host proteins. Here, we show that these anti-CRISPR proteins display diverse inhibition mechanisms through accurate structural prediction and functional analysis. We find that these phage-derived proteins are extremely distinct in structure, some of which have no homologues in the current protein structure domain. Furthermore, we find a novel family of anti-CRISPR proteins which are structurally similar to the recently discovered mechanism of manipulating host proteins through enzymatic activity, rather than through direct inference. Using highly accurate structure prediction, we present a wide variety of protein-manipulating strategies of anti-CRISPR proteins for future protein drug design.}, }
@article {pmid35336126, year = {2022}, author = {de Oliveira, IMF and Godoy-Santos, F and Oyama, LB and Moreira, SM and Dias, RG and Huws, SA and Creevey, CJ and Mantovani, HC}, title = {Whole-Genome Sequencing and Comparative Genomic Analysis of Antimicrobial Producing Streptococcus lutetiensis from the Rumen.}, journal = {Microorganisms}, volume = {10}, number = {3}, pages = {}, pmid = {35336126}, issn = {2076-2607}, support = {001//Coordenação de Aperfeicoamento de Pessoal de Nível Superior/ ; APQ-02899-18//Fundação de Amparo à Pesquisa do Estado de Minas Gerais/ ; //National Council for Scientific and Technological Development/ ; #CNPq//Institutos Nacionais de Ciência e Tecnologia/ ; 172629373//RCUK Newton Institutional Link Funding/ ; }, abstract = {Antimicrobial peptides (AMPs) can efficiently control different microbial pathogens and show the potential to be applied in clinical practice and livestock production. In this work, the aim was to isolate AMP-producing ruminal streptococci and to characterize their genetic features through whole-genome sequencing. We cultured 463 bacterial isolates from the rumen of Nelore bulls, 81 of which were phenotypically classified as being Streptococcaceae. Five isolates with broad-range activity were genome sequenced and confirmed as being Streptococcus lutetiensis. The genetic features linked to their antimicrobial activity or adaptation to the rumen environment were characterized through comparative genomics. The genome of S. lutetiensis UFV80 harbored a putative CRISPR-Cas9 system (Type IIA). Computational tools were used to discover novel biosynthetic clusters linked to the production of bacteriocins. All bacterial genomes harbored genetic clusters related to the biosynthesis of class I and class II bacteriocins. SDS-PAGE confirmed the results obtained in silico and demonstrated that the class II bacteriocins predicted in the genomes of three S. lutetiensis strains had identical molecular mass (5197 Da). These results demonstrate that ruminal bacteria of the Streptococcus bovis/equinus complex represent a promising source of novel antimicrobial peptides.}, }
@article {pmid35335122, year = {2022}, author = {Jiang, Z and Abdullah,  and Zhang, S and Jiang, Y and Liu, R and Xiao, Y}, title = {Development and Optimization of CRISPR Prime Editing System in Photoautotrophic Cells.}, journal = {Molecules (Basel, Switzerland)}, volume = {27}, number = {6}, pages = {}, pmid = {35335122}, issn = {1420-3049}, support = {2018YFA0901200; 31870071; SL2020MS028//National Key R&amp;D Program of China; National Natural Science Foundation of China; Oceanic Interdisciplinary Program of Shanghai Jiao Tong University/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; INDEL Mutation ; Point Mutation ; RNA, Guide/genetics ; }, abstract = {Prime editor (PE), a versatile editor that allows the insertion and deletion of arbitrary sequences, and all 12-point mutations without double-strand breaks (DSB) and a donor template, dramatically enhances research capabilities. PE combines nickase Cas9(H840A) and reverse transcriptase (RT), along with prime editing guide RNA (pegRNA). It has been reported in several plant species, but a weak editing efficiency has led to a decrease in applications. This study reports an optimized-prime editor (O-PE) for endogenous gene editing in Arabidopsis thaliana cells, with an average 1.15% editing efficiency, which is 16.4-fold higher than previously reported. Meanwhile, we observed an increase in indels when testing alternative reverse transcriptase and found out that nCas9(H840A) fused to non-functional reverse transcriptase was responsible for the increase. This work develops an efficient prime editor for plant cells and provides a blueprint for applying PE in other photoautotrophic cells, such as microalgae, that have a high industrial value.}, }
@article {pmid35333864, year = {2022}, author = {Zhang, Q and Zhang, Y and Chai, Y}, title = {Optimization of CRISPR/LbCas12a-mediated gene editing in Arabidopsis.}, journal = {PloS one}, volume = {17}, number = {3}, pages = {e0265114}, pmid = {35333864}, issn = {1932-6203}, mesh = {*Arabidopsis/genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing/methods ; Promoter Regions, Genetic ; }, abstract = {CRISPR/LbCas12a system (LbCpf1) has been widely used for genome modification including plant species. However, the efficiency of CRISPR/LbCas12a varied considerably in different plant species and tissues, and the editing efficiency needs to be further improved. In this study, we tried to improve the editing efficiency of CRISPR/LbCas12a in Arabidopsis by optimizing the crRNA expression strategies and Pol II promoters. Notably, the combination of tRNA-crRNA fusion strategy and RPS5A promoter in CRISPR/LbCas12a system has highest editing efficiency, while CRISPR/LbCas12a driven by EC1f-in(crR)p had the highest ratio of homozygous &amp; bi-allelic mutants. In addition, all homozygous &amp; bi-allelic mutants can be stably inherited to the next generation and have no phenotypic separation. In this study, the editing efficiency of the CRISPR/LbCas12a system was improved by selecting the optimal crRNA expression strategies and promoter of LbCas12a in Arabidopsis, which will prove useful for optimization of CRISPR/LbCas12a methods in other plants.}, }
@article {pmid35333620, year = {2022}, author = {Juríková, K and Sepšiová, R and Ševčovičová, A and Tomáška, Ľ and Džugasová, V}, title = {Implementing CRISPR-Cas9 Yeast Practicals into Biology Curricula.}, journal = {The CRISPR journal}, volume = {5}, number = {2}, pages = {181-186}, doi = {10.1089/crispr.2021.0125}, pmid = {35333620}, issn = {2573-1602}, mesh = {Biology ; *CRISPR-Cas Systems/genetics ; Curriculum ; Gene Editing/methods ; Humans ; *Saccharomyces cerevisiae/genetics ; }, abstract = {CRISPR-Cas9 is a genome-editing technique that has been widely adopted thanks to its simplicity, efficiency, and broad application potential. Due to its advantages and pervasive use, there have been attempts to include this method in the existing curricula for students majoring in various disciplines of biology. In this perspective, we summarize the existing CRISPR-Cas courses that harness a well-established model organism: baker's yeast, Saccharomyces cerevisiae. As an example, we present a detailed description of a fully hands-on, flexible, robust, and cost-efficient practical CRISPR-Cas9 course, where students participate in yeast genome editing at every stage-from the bioinformatic design of single-guide RNA, through molecular cloning and yeast transformation, to the final confirmation of the introduced mutation. Finally, we emphasize that in addition to providing experimental skills and theoretical knowledge, the practical courses on CRISPR-Cas represent ideal platforms for discussing the ethical implications of the democratization of biology.}, }
@article {pmid35333175, year = {2022}, author = {Thumberger, T and Tavhelidse-Suck, T and Gutierrez-Triana, JA and Cornean, A and Medert, R and Welz, B and Freichel, M and Wittbrodt, J}, title = {Boosting targeted genome editing using the hei-tag.}, journal = {eLife}, volume = {11}, number = {}, pages = {}, pmid = {35333175}, issn = {2050-084X}, support = {CRC873,project A3//Deutsche Forschungsgemeinschaft/ ; FOR2509 P10,WI 1824/9-1//Deutsche Forschungsgemeinschaft/ ; CRC1118,project S03//Deutsche Forschungsgemeinschaft/ ; GA 294354-ManISteC//H2020 European Research Council/ ; NO 810172//ERC-SyG H2020/ ; 3DMM2O, EXC 2082/1 Wittbrodt C3//Deutsche Forschungsgemeinschaft/ ; CRC873 project A3//Deutsche Forschungsgemeinschaft/ ; FOR2509 P10 WI 1824/9-1//Deutsche Forschungsgemeinschaft/ ; CRC1118 project S03//Deutsche Forschungsgemeinschaft/ ; 810172//H2020 European Research Council/ ; }, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Cytosine ; *Gene Editing/methods ; Mammals ; Nuclear Localization Signals ; RNA, Messenger/genetics ; }, abstract = {Precise, targeted genome editing by CRISPR/Cas9 is key for basic research and translational approaches in model and non-model systems. While active in all species tested so far, editing efficiencies still leave room for improvement. The bacterial Cas9 needs to be efficiently shuttled into the nucleus as attempted by fusion with nuclear localization signals (NLSs). Additional peptide tags such as FLAG- or myc-tags are usually added for immediate detection or straightforward purification. Immediate activity is usually granted by administration of preassembled protein/RNA complexes. We present the 'hei-tag (high efficiency-tag)' which boosts the activity of CRISPR/Cas genome editing tools already when supplied as mRNA. The addition of the hei-tag, a myc-tag coupled to an optimized NLS via a flexible linker, to Cas9 or a C-to-T (cytosine-to-thymine) base editor dramatically enhances the respective targeting efficiency. This results in an increase in bi-allelic editing, yet reduction of allele variance, indicating an immediate activity even at early developmental stages. The hei-tag boost is active in model systems ranging from fish to mammals, including tissue culture applications. The simple addition of the hei-tag allows to instantly upgrade existing and potentially highly adapted systems as well as to establish novel highly efficient tools immediately applicable at the mRNA level.}, }
@article {pmid35332696, year = {2022}, author = {Aragonés, V and Aliaga, F and Pasin, F and Daròs, JA}, title = {Simplifying plant gene silencing and genome editing logistics by a one-Agrobacterium system for simultaneous delivery of multipartite virus vectors.}, journal = {Biotechnology journal}, volume = {}, number = {}, pages = {e2100504}, doi = {10.1002/biot.202100504}, pmid = {35332696}, issn = {1860-7314}, abstract = {Viral vectors provide a quick and effective way to express exogenous sequences in eukaryotic cells and to engineer eukaryotic genomes through the delivery of CRISPR/Cas components. Here, we presentJoinTRV, an improved vector system based on tobacco rattle virus (TRV) that simplifies gene silencing and genome editing logistics. Our system consists of two mini T-DNA vectors from which TRV RNA1 (pLX-TRV1) and an engineered version of TRV RNA2 (pLX-TRV2) are expressed. The two vectors have compatible origins that allow their cotransformation and maintenance into a single Agrobacterium cell, as well as their simultaneous delivery to plants by a one-Agrobacterium/two-vector approach. The JoinTRV vectors are substantially smaller than those of any known TRV vector system, and pLX-TRV2 can be easily customized to express desired sequences by one-step digestion-ligation and homology-based cloning. The system was successfully used in Nicotiana benthamiana for launching TRV infection, for recombinant protein production, as well as for robust virus-induced gene silencing (VIGS) of endogenous transcripts using bacterial suspensions at low optical densities. JoinTRV-mediated delivery of single-guide RNAs in a Cas9 transgenic host allowed somatic cell editing efficiencies of ≈90%; editing events were heritable and >50% of the progeny seedlings showed mutations at the targeted loci.}, }
@article {pmid35332138, year = {2022}, author = {Peterka, M and Akrap, N and Li, S and Wimberger, S and Hsieh, PP and Degtev, D and Bestas, B and Barr, J and van de Plassche, S and Mendoza-Garcia, P and Šviković, S and Sienski, G and Firth, M and Maresca, M}, title = {Harnessing DSB repair to promote efficient homology-dependent and -independent prime editing.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {1240}, pmid = {35332138}, issn = {2041-1723}, mesh = {Animals ; CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; *DNA End-Joining Repair ; DNA Repair ; Endonucleases/metabolism ; Gene Editing ; Mammals/genetics ; }, abstract = {Prime editing recently emerged as a next-generation approach for precise genome editing. Here we exploit DNA double-strand break (DSB) repair to develop two strategies that install precise genomic insertions using an SpCas9 nuclease-based prime editor (PEn). We first demonstrate that PEn coupled to a regular prime editing guide RNA (pegRNA) efficiently promotes short genomic insertions through a homology-dependent DSB repair mechanism. While PEn editing leads to increased levels of by-products, it can rescue pegRNAs that perform poorly with a nickase-based prime editor. We also present a small molecule approach that yields increased product purity of PEn editing. Next, we develop a homology-independent PEn editing strategy, which installs genomic insertions at DSBs through the non-homologous end joining pathway (NHEJ). Lastly, we show that PEn-mediated insertions at DSBs prevent Cas9-induced large chromosomal deletions and provide evidence that continuous Cas9-mediated cutting is one of the mechanisms by which Cas9-induced large deletions arise. Altogether, this work expands the current prime editing toolbox by leveraging distinct DNA repair mechanisms including NHEJ, which represents the primary pathway of DSB repair in mammalian cells.}, }
@article {pmid35331236, year = {2022}, author = {Selvakumar, SC and Preethi, KA and Ross, K and Tusubira, D and Khan, MWA and Mani, P and Rao, TN and Sekar, D}, title = {CRISPR/Cas9 and next generation sequencing in the personalized treatment of Cancer.}, journal = {Molecular cancer}, volume = {21}, number = {1}, pages = {83}, pmid = {35331236}, issn = {1476-4598}, mesh = {CRISPR-Cas Systems ; Gene Editing/methods ; High-Throughput Nucleotide Sequencing ; Humans ; *Neoplasms/genetics/therapy ; *Precision Medicine ; }, abstract = {BACKGROUND: Cancer is caused by a combination of genetic and epigenetic abnormalities. Current cancer therapies are limited due to the complexity of their mechanism, underlining the need for alternative therapeutic approaches. Interestingly, combining the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) system with next-generation sequencing (NGS) has the potential to speed up the identification, validation, and targeting of high-value targets.<br><br>MAIN TEXT: Personalized or precision medicine combines genetic information with phenotypic and environmental characteristics to produce healthcare tailored to the individual and eliminates the constraints of "one-size-fits-all" therapy. Precision medicine is now possible thanks to cancer genome sequencing. Having advantages over limited sample requirements and the recent development of biomarkers have made the use of NGS a major leap in personalized medicine. Tumor and cell-free DNA profiling using NGS, proteome and RNA analyses, and a better understanding of immunological systems, are all helping to improve cancer treatment choices. Finally, direct targeting of tumor genes in cancer cells with CRISPR/Cas9 may be achievable, allowing for eliminating genetic changes that lead to tumor growth and metastatic capability.<br><br>CONCLUSION: With NGS and CRISPR/Cas9, the goal is no longer to match the treatment for the diagnosed tumor but rather to build a treatment method that fits the tumor exactly. Hence, in this review, we have discussed the potential role of CRISPR/Cas9 and NGS in advancing personalized medicine.}, }
@article {pmid35331211, year = {2022}, author = {Garcia-Perez, E and Diego-Martin, B and Quijano-Rubio, A and Moreno-Giménez, E and Selma, S and Orzaez, D and Vazquez-Vilar, M}, title = {A copper switch for inducing CRISPR/Cas9-based transcriptional activation tightly regulates gene expression in Nicotiana benthamiana.}, journal = {BMC biotechnology}, volume = {22}, number = {1}, pages = {12}, pmid = {35331211}, issn = {1472-6750}, mesh = {*CRISPR-Cas Systems/genetics ; Copper ; Gene Expression ; Plants/genetics ; *Tobacco/genetics ; Transcriptional Activation ; }, abstract = {BACKGROUND: CRISPR-based programmable transcriptional activators (PTAs) are used in plants for rewiring gene networks. Better tuning of their activity in a time and dose-dependent manner should allow precise control of gene expression. Here, we report the optimization of a Copper Inducible system called CI-switch for conditional gene activation in Nicotiana benthamiana. In the presence of copper, the copper-responsive factor CUP2 undergoes a conformational change and binds a DNA motif named copper-binding site (CBS).<br><br>RESULTS: In this study, we tested several activation domains fused to CUP2 and found that the non-viral Gal4 domain results in strong activation of a reporter gene equipped with a minimal promoter, offering advantages over previous designs. To connect copper regulation with downstream programmable elements, several copper-dependent configurations of the strong dCasEV2.1 PTA were assayed, aiming at maximizing activation range, while minimizing undesired background expression. The best configuration involved a dual copper regulation of the two protein components of the PTA, namely dCas9:EDLL and MS2:VPR, and a constitutive RNA pol III-driven expression of the third component, a guide RNA with anchoring sites for the MS2 RNA-binding domain. With these optimizations, the CI/dCasEV2.1 system resulted in copper-dependent activation rates of 2,600-fold and 245-fold for the endogenous N. benthamiana DFR and PAL2 genes, respectively, with negligible expression in the absence of the trigger.<br><br>CONCLUSIONS: The tight regulation of copper over CI/dCasEV2.1 makes this system ideal for the conditional production of plant-derived metabolites and recombinant proteins in the field.}, }
@article {pmid35331142, year = {2022}, author = {Blomme, J and Develtere, W and Köse, A and Arraiza Ribera, J and Brugmans, C and Jaraba-Wallace, J and Decaestecker, W and Rombaut, D and Baekelandt, A and Daniel Fernández Fernández, Á and Van Breusegem, F and Inzé, D and Jacobs, T}, title = {The heat is on: a simple method to increase genome editing efficiency in plants.}, journal = {BMC plant biology}, volume = {22}, number = {1}, pages = {142}, pmid = {35331142}, issn = {1471-2229}, support = {12T3418N//Fonds Wetenschappelijk Onderzoek/ ; 3G038719//Fonds Wetenschappelijk Onderzoek/ ; BOF20/PDO/016//Bijzonder Onderzoeksfonds UGent/ ; BOFMET2015000201//Bijzonder Onderzoeksfonds UGent/ ; }, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Genome, Plant/genetics ; Mutagenesis ; Plants, Genetically Modified/genetics ; }, abstract = {BACKGROUND: Precision genome mutagenesis using CRISPR/Cas has become the standard method to generate mutant plant lines. Several improvements have been made to increase mutagenesis efficiency, either through vector optimisation or the application of heat stress.<br><br>RESULTS: Here, we present a simplified heat stress assay that can be completed in six days using commonly-available laboratory equipment. We show that three heat shocks (3xHS) efficiently increases indel efficiency of LbCas12a and Cas9, irrespective of the target sequence or the promoter used to express the nuclease. The generated indels are primarily somatic, but for three out of five targets we demonstrate that up to 25% more biallelic mutations are transmitted to the progeny when heat is applied compared to non-heat controls. We also applied our heat treatment to lines containing CRISPR base editors and observed a 22-27% increase in the percentage of C-to-T base editing. Furthermore, we test the effect of 3xHS on generating large deletions and a homologous recombination reporter. Interestingly, we observed no positive effect of 3xHS treatment on either approach using our conditions.<br><br>CONCLUSIONS: Together, our experiments show that heat treatment is consistently effective at increasing the number of somatic mutations using many CRISPR approaches in plants and in some cases can increase the recovery of mutant progeny.}, }
@article {pmid35330963, year = {2022}, author = {Angelopoulou, A and Papaspyropoulos, A and Papantonis, A and Gorgoulis, VG}, title = {CRISPR-Cas9-mediated induction of large chromosomal inversions in human bronchial epithelial cells.}, journal = {STAR protocols}, volume = {3}, number = {2}, pages = {101257}, pmid = {35330963}, issn = {2666-1667}, mesh = {*CRISPR-Cas Systems/genetics ; Cells, Cultured ; *Chromosome Inversion ; Epithelial Cells ; Gene Editing/methods ; Humans ; }, abstract = {The in vitro recapitulation of chromosomal rearrangements is a necessary tool for understanding malignancy at the molecular level. Here, we describe the targeted induction of a large chromosomal inversion (>3.7 Mbp) through CRISPR-Cas9-mediated genome editing. As inversions occur at low frequency following Cas9 cleavage, we provide a detailed screening approach of FACS-sorted, single-cell-derived clonal human bronchial epithelial cell (HBEC) cultures. The protocol provided is tailored to HBECs; however, it can be readily applied to additional adherent cellular models. For complete details on the use and execution of this protocol, please refer to Zampetidis et al. (2021).}, }
@article {pmid35330692, year = {2022}, author = {Sharma, P and Lew, TTS}, title = {Principles of Nanoparticle Design for Genome Editing in Plants.}, journal = {Frontiers in genome editing}, volume = {4}, number = {}, pages = {846624}, pmid = {35330692}, issn = {2673-3439}, abstract = {Precise plant genome editing technologies have provided new opportunities to accelerate crop improvement and develop more sustainable agricultural systems. In particular, the prokaryote-derived CRISPR platforms allow precise manipulation of the crop genome, enabling the generation of high-yielding and stress-tolerant crop varieties. Nanotechnology has the potential to catalyze the development of a novel molecular toolbox even further by introducing the possibility of a rapid, universal delivery method to edit the plant genome in a species-independent manner. In this Perspective, we highlight how nanoparticles can help unlock the full potential of CRISPR/Cas technology in targeted manipulation of the plant genome to improve agricultural output. We discuss current challenges hampering progress in nanoparticle-enabled plant gene-editing research and application in the field, and highlight how rational nanoparticle design can overcome them. Finally, we examine the implications of the regulatory frameworks and social acceptance for the future of nano-enabled precision breeding in the developing world.}, }
@article {pmid35330117, year = {2022}, author = {Zakharevich, NV and Nikitin, MS and Kovtun, AS and Malov, VO and Averina, OV and Danilenko, VN and Artamonova, II}, title = {CRISPR-Cas Systems in Gut Microbiome of Children with Autism Spectrum Disorders.}, journal = {Life (Basel, Switzerland)}, volume = {12}, number = {3}, pages = {}, pmid = {35330117}, issn = {2075-1729}, support = {18-29-07087//Russian Foundation for Basic Research/ ; }, abstract = {The human gut microbiome is associated with various diseases, including autism spectrum disorders (ASD). Variations of the taxonomical composition in the gut microbiome of children with ASD have been observed repeatedly. However, features and parameters of the microbiome CRISPR-Cas systems in ASD have not been investigated yet. Here, we demonstrate such an analysis in order to describe the overall changes in the microbiome CRISPR-Cas systems during ASD as well as to reveal their potential to be used in diagnostics and therapy. For the systems identification, we used a combination of the publicly available tools suited for completed genomes with subsequent filtrations. In the considered data, the microbiomes of children with ASD contained fewer arrays per Gb of assembly than the control group, but the arrays included more spacers on average. CRISPR arrays from the microbiomes of children with ASD differed from the control group neither in the fractions of spacers with protospacers from known genomes, nor in the sets of known bacteriophages providing protospacers. Almost all bacterial protospacers of the gut microbiome systems for both children with ASD and the healthy ones were located in prophage islands, leaving no room for the systems to participate in the interspecies competition.}, }
@article {pmid35328599, year = {2022}, author = {Mészár, Z and Kókai, É and Varga, R and Ducza, L and Papp, T and Béresová, M and Nagy, M and Szücs, P and Varga, A}, title = {CRISPR/Cas9-Based Mutagenesis of Histone H3.1 in Spinal Dynorphinergic Neurons Attenuates Thermal Sensitivity in Mice.}, journal = {International journal of molecular sciences}, volume = {23}, number = {6}, pages = {}, pmid = {35328599}, issn = {1422-0067}, support = {FK 125035//National Research, Development and Innovation Office/ ; BO/00369/17/5//János Bolyai Scholarship of the Hungarian Academy of Sciences/ ; ÚNKP-19-4-DE-3//New National Excelence Program of the Ministry of Human Capacities/ ; 2017-1.2.1-NKP-2017-00002//Hungarian Brain Research Program/ ; }, mesh = {Animals ; *Burns/genetics ; CRISPR-Cas Systems/genetics ; *Histones/genetics/metabolism ; Hyperalgesia/metabolism ; Mice ; Mutagenesis ; Neurons/metabolism ; Spinal Cord/metabolism ; }, abstract = {Burn injury is a trauma resulting in tissue degradation and severe pain, which is processed first by neuronal circuits in the spinal dorsal horn. We have recently shown that in mice, excitatory dynorphinergic (Pdyn) neurons play a pivotal role in the response to burn-injury-associated tissue damage via histone H3.1 phosphorylation-dependent signaling. As Pdyn neurons were mostly associated with mechanical allodynia, their involvement in thermonociception had to be further elucidated. Using a custom-made AAV9_mutH3.1 virus combined with the CRISPR/cas9 system, here we provide evidence that blocking histone H3.1 phosphorylation at position serine 10 (S10) in spinal Pdyn neurons significantly increases the thermal nociceptive threshold in mice. In contrast, neither mechanosensation nor acute chemonociception was affected by the transgenic manipulation of histone H3.1. These results suggest that blocking rapid epigenetic tagging of S10H3 in spinal Pdyn neurons alters acute thermosensation and thus explains the involvement of Pdyn cells in the immediate response to burn-injury-associated tissue damage.}, }
@article {pmid35328559, year = {2022}, author = {Aulitto, M and Martinez-Alvarez, L and Fiorentino, G and Limauro, D and Peng, X and Contursi, P}, title = {A Comparative Analysis of Weizmannia coagulans Genomes Unravels the Genetic Potential for Biotechnological Applications.}, journal = {International journal of molecular sciences}, volume = {23}, number = {6}, pages = {}, pmid = {35328559}, issn = {1422-0067}, support = {E69E19000530001//Ministero dell'Università e della Ricerca/ ; 2017-JTNK78.006//Ministero dell'Università e della Ricerca/ ; NNF17OC0031154//Novo Nordisk (Denmark)/ ; }, mesh = {Base Sequence ; CRISPR-Cas Systems/genetics ; *Genome, Bacterial ; *Genomics ; }, abstract = {The production of biochemicals requires the use of microbial strains with efficient substrate conversion and excellent environmental robustness, such as Weizmannia coagulans species. So far, the genomes of 47 strains have been sequenced. Herein, we report a comparative genomic analysis of nine strains on the full repertoire of Carbohydrate-Active enZymes (CAZymes), secretion systems, and resistance mechanisms to environmental challenges. Moreover, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) immune system along with CRISPR-associated (Cas) genes, was also analyzed. Overall, this study expands our understanding of the strain's genomic diversity of W. coagulans to fully exploit its potential in biotechnological applications.}, }
@article {pmid35328421, year = {2022}, author = {Porika, M and Tippani, R and Saretzki, GC}, title = {CRISPR/Cas: A New Tool in the Research of Telomeres and Telomerase as Well as a Novel Form of Cancer Therapy.}, journal = {International journal of molecular sciences}, volume = {23}, number = {6}, pages = {}, pmid = {35328421}, issn = {1422-0067}, mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Neoplasms/genetics/therapy ; *Telomerase/genetics/metabolism ; Telomere/genetics/metabolism ; }, abstract = {Due to their close connection with senescence, aging, and disease, telomeres and telomerase provide a unique and vital research route for boosting longevity and health span. Despite significant advances during the last three decades, earlier studies into these two biological players were impeded by the difficulty of achieving real-time changes inside living cells. As a result of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated system's (Cas) method, targeted genetic studies are now underway to change telomerase, the genes that govern it as well as telomeres. This review will discuss studies that have utilized CRISPR-related technologies to target and modify genes relevant to telomeres and telomerase as well as to develop targeted anti-cancer therapies. These studies greatly improve our knowledge and understanding of cellular and molecular mechanisms that underlie cancer development and aging.}, }
@article {pmid35327601, year = {2022}, author = {Li, B and Ai, D and Liu, X}, title = {CNN-XG: A Hybrid Framework for sgRNA On-Target Prediction.}, journal = {Biomolecules}, volume = {12}, number = {3}, pages = {}, pmid = {35327601}, issn = {2218-273X}, mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Neural Networks, Computer ; *RNA, Guide/genetics/metabolism ; }, abstract = {As the third generation gene editing technology, Crispr/Cas9 has a wide range of applications. The success of Crispr depends on the editing of the target gene via a functional complex of sgRNA and Cas9 proteins. Therefore, highly specific and high on-target cleavage efficiency sgRNA can make this process more accurate and efficient. Although there are already many sophisticated machine learning or deep learning models to predict the on-target cleavage efficiency of sgRNA, prediction accuracy remains to be improved. XGBoost is good at classification as the ensemble model could overcome the deficiency of a single classifier to classify, and we would like to improve the prediction efficiency for sgRNA on-target activity by introducing XGBoost into the model. We present a novel machine learning framework which combines a convolutional neural network (CNN) and XGBoost to predict sgRNA on-target knockout efficacy. Our framework, called CNN-XG, is mainly composed of two parts: a feature extractor CNN is used to automatically extract features from sequences and predictor XGBoost is applied to predict features extracted after convolution. Experiments on commonly used datasets show that CNN-XG performed significantly better than other existing frameworks in the predicted classification mode.}, }
@article {pmid35326449, year = {2022}, author = {Jamehdor, S and Pajouhanfar, S and Saba, S and Uzan, G and Teimoori, A and Naserian, S}, title = {Principles and Applications of CRISPR Toolkit in Virus Manipulation, Diagnosis, and Virus-Host Interactions.}, journal = {Cells}, volume = {11}, number = {6}, pages = {}, pmid = {35326449}, issn = {2073-4409}, mesh = {*COVID-19/diagnosis ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA Viruses ; Host Microbial Interactions ; Humans ; SARS-CoV-2/genetics ; *Virus Diseases/diagnosis/genetics ; *Viruses/genetics ; }, abstract = {Viruses are one of the most important concerns for human health, and overcoming viral infections is a worldwide challenge. However, researchers have been trying to manipulate viral genomes to overcome various disorders, including cancer, for vaccine development purposes. CRISPR (clustered regularly interspaced short palindromic repeats) is becoming one of the most functional and widely used tools for RNA and DNA manipulation in multiple organisms. This approach has provided an unprecedented opportunity for creating simple, inexpensive, specific, targeted, accurate, and practical manipulations of viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus-1 (HIV-1), and vaccinia virus. Furthermore, this method can be used to make an effective and precise diagnosis of viral infections. Nevertheless, a valid and scientifically designed CRISPR system is critical to make more effective and accurate changes in viruses. In this review, we have focused on the best and the most effective ways to design sgRNA, gene knock-in(s), and gene knock-out(s) for virus-targeted manipulation. Furthermore, we have emphasized the application of CRISPR technology in virus diagnosis and in finding significant genes involved in virus-host interactions.}, }
@article {pmid35325722, year = {2022}, author = {Zhuang, J and Zhao, Z and Lian, K and Yin, L and Wang, J and Man, S and Liu, G and Ma, L}, title = {SERS-based CRISPR/Cas assay on microfluidic paper analytical devices for supersensitive detection of pathogenic bacteria in foods.}, journal = {Biosensors &amp; bioelectronics}, volume = {207}, number = {}, pages = {114167}, doi = {10.1016/j.bios.2022.114167}, pmid = {35325722}, issn = {1873-4235}, mesh = {Bacteria/genetics ; *Biosensing Techniques ; *CRISPR-Cas Systems ; Microfluidics ; Recombinases ; Salmonella typhimurium/genetics ; Spectrum Analysis, Raman ; }, abstract = {Rapid and point-of-need (PON) detection of bacteria is crucial to directly provide rapid and reliable diagnostics information during on-site tests, allowing more room for taking proactive measures. By taking the multifaceted advantages of CRISPR/Cas12a and surface-enhanced Raman scattering (SERS), for the first time, we designed a recombinase polymerase amplification (RPA)-integrated microfluidic paper-based analytical device (μPAD), coined RPA-Cas12a-μPAD for supersensitive SERS detection. Single-stranded DNAs were designed to "pull down" SERS nanoprobes. The amplicons of the invA gene triggered the trans-cleavage of Cas12a, resulting in the indiscriminate shredding of linker ssDNA. Thus, the degree of aggregation of SERS nanoprobes was dependent on the concentration of Salmonella typhimurium (S. typhi), which was determined on a μPAD and monitored by a Raman spectrometer. The limit of detection for S. typhi was approximately 3-4 CFU/mL for spiked milk and meat samples with a dynamic detection range from 1 to 108 CFU/mL. The RPA-Cas12a-μPAD secured accurate tests for food samples in 45 min. This work expands the reach of CRISPR-based diagnostics (CRISPR-Dx) and provides a novel and robust bacterial PON detection platform.}, }
@article {pmid35325565, year = {2022}, author = {Huerne, K and Palmour, N and Wu, AR and Beck, S and Berner, A and Siebert, R and Joly, Y}, title = {Auditing the Editor: A Review of Key Translational Issues in Epigenetic Editing.}, journal = {The CRISPR journal}, volume = {5}, number = {2}, pages = {203-212}, doi = {10.1089/crispr.2021.0094}, pmid = {35325565}, issn = {2573-1602}, support = {CEE-151618//CIHR/Canada ; }, mesh = {*CRISPR-Cas Systems/genetics ; Epigenomics ; *Gene Editing ; Humans ; }, abstract = {Currently, most advances in site-specific epigenetic editing for human use are concentrated in basic research, yet, there is considerable interest to translate this technology beyond the bench. This review highlights recent developments with epigenetic editing technology in comparison with the canonical CRISPR-Cas genome editing, as well as the epistemic and ethical considerations with preemptive translation of epigenetic editing into clinical or commercial use in humans. Key considerations in safety, equity, and access to epigenetic editing are highlighted, with a spotlight on the ethical, legal, and social issues of this technology in the context of global health equity.}, }
@article {pmid35325432, year = {2022}, author = {Movahedi, A and Hajiahmadi, Z and Wei, H and Yang, L and Ruan, H and Zhuge, Q}, title = {A Method to Reduce off-Targets in CRISPR/Cas9 System in Plants.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2408}, number = {}, pages = {317-324}, pmid = {35325432}, issn = {1940-6029}, mesh = {*CRISPR-Cas Systems/genetics ; Mutagenesis ; Mutation ; Plasmids/genetics ; *Silicon Dioxide ; }, abstract = {One of the strategies to reduce the off-target mutations in CRISPR/Cas9 system is to use the temperature-independent gene transformation method. Mesoporous silica nanoparticles (MSNs)-gene delivery system is temperature-independent; thus, it can transfer the interesting plasmid (pDNA) to the target plant at different temperatures, including 37 °C. Due to the high activity of SpCas9 at 37 °C compared to lower temperatures, on-target mutagenesis increases at 37 °C. Therefore, we describe the synthesis of the functionalized MSNs with the particle size of less than 40 nm, binding pDNA to the MSNs, and transferring of the pDNA-MSNs into the target plants.}, }
@article {pmid35325413, year = {2022}, author = {Pandey, P and Mysore, KS and Senthil-Kumar, M}, title = {Recent Advances in Plant Gene Silencing Methods.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2408}, number = {}, pages = {1-22}, pmid = {35325413}, issn = {1940-6029}, mesh = {Gene Editing ; *Gene Silencing ; Genes, Plant ; *Plants/genetics ; }, abstract = {With the increasing understanding of fundamentals of gene silencing pathways in plants, various tools and techniques for downregulating the expression of a target gene have been developed across multiple plant species. This chapter provides an insight into the molecular mechanisms of gene silencing and highlights the advancements in various gene silencing approaches. The prominent aspects of different gene silencing methods, their advantages and disadvantages have been discussed. A succinct discussion on the newly emerged microRNA-based technologies like microRNA-induced gene silencing (MIGS) and microRNA-mediated virus-induced gene silencing (MIR-VIGS) are also presented. We have also discussed the gene-editing system like CRISPR-Cas. The prominent bottlenecks in gene silencing methods are the off-target effects and lack of universal applicability. However, the tremendous growth in understanding of this field reflects the potentials for improvements in the currently available approaches and the development of new widely applicable methods for easy, fast, and efficient functional characterization of plant genes.}, }
@article {pmid35325044, year = {2022}, author = {Gladka, MM and Baker, AH}, title = {Cutting a path to effective delivery of genome engineering machinery.}, journal = {Cardiovascular research}, volume = {118}, number = {6}, pages = {e42-e44}, doi = {10.1093/cvr/cvac034}, pmid = {35325044}, issn = {1755-3245}, support = {NHS2020T041//Dekker Senior Scientist fellowship from the Dutch Heart Foundation/ ; //British Heart Foundation Chair of Translational Cardiovascular Sciences/ ; }, mesh = {*CRISPR-Cas Systems ; *Gene Editing ; }, }
@article {pmid35323947, year = {2022}, author = {Liang, M and Liu, L and Xu, F and Zeng, X and Wang, R and Yang, J and Wang, W and Karthik, L and Liu, J and Yang, Z and Zhu, G and Wang, S and Bai, L and Tong, Y and Liu, X and Wu, M and Zhang, LX and Tan, GY}, title = {Activating cryptic biosynthetic gene cluster through a CRISPR-Cas12a-mediated direct cloning approach.}, journal = {Nucleic acids research}, volume = {50}, number = {6}, pages = {3581-3592}, pmid = {35323947}, issn = {1362-4962}, mesh = {*Biological Products ; CRISPR-Cas Systems ; Cloning, Molecular ; Multigene Family ; *Streptomyces/genetics ; }, abstract = {Direct cloning of biosynthetic gene clusters (BGCs) from microbial genomes facilitates natural product-based drug discovery. Here, by combining Cas12a and the advanced features of bacterial artificial chromosome library construction, we developed a fast yet efficient in vitro platform for directly capturing large BGCs, named CAT-FISHING (CRISPR/Cas12a-mediated fast direct biosynthetic gene cluster cloning). As demonstrations, several large BGCs from different actinomycetal genomic DNA samples were efficiently captured by CAT-FISHING, the largest of which was 145 kb with 75% GC content. Furthermore, the directly cloned, 110 kb long, cryptic polyketide encoding BGC from Micromonospora sp. 181 was then heterologously expressed in a Streptomyces chassis. It turned out to be a new macrolactam compound, marinolactam A, which showed promising anticancer activity. Our results indicate that CAT-FISHING is a powerful method for complicated BGC cloning, and we believe that it would be an important asset to the entire community of natural product-based drug discovery.}, }
@article {pmid35323942, year = {2022}, author = {Yoo, KW and Yadav, MK and Song, Q and Atala, A and Lu, B}, title = {Targeting DNA polymerase to DNA double-strand breaks reduces DNA deletion size and increases templated insertions generated by CRISPR/Cas9.}, journal = {Nucleic acids research}, volume = {50}, number = {7}, pages = {3944-3957}, pmid = {35323942}, issn = {1362-4962}, mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; DNA/genetics/metabolism ; DNA-Directed DNA Polymerase/metabolism ; *Gene Editing/methods ; }, abstract = {Most insertions or deletions generated by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) endonucleases are short (<25 bp), but unpredictable on-target long DNA deletions (>500 bp) can be observed. The possibility of generating long on-target DNA deletions poses safety risks to somatic genome editing and makes the outcomes of genome editing less predictable. Methods for generating refined mutations are desirable but currently unavailable. Here, we show that fusing Escherichia coli DNA polymerase I or the Klenow fragment to Cas9 greatly increases the frequencies of 1-bp deletions and decreases >1-bp deletions or insertions. Importantly, doing so also greatly decreases the generation of long deletions, including those >2 kb. In addition, templated insertions (the insertion of the nucleotide 4 nt upstream of the protospacer adjacent motif) were increased relative to other insertions. Counteracting DNA resection was one of the mechanisms perturbing deletion sizes. Targeting DNA polymerase to double-strand breaks did not increase off-targets or base substitution rates around the cleavage sites, yet increased editing efficiency in primary cells. Our strategy makes it possible to generate refined DNA mutations for improved safety without sacrificing efficiency of genome editing.}, }
@article {pmid35323424, year = {2022}, author = {Li, C and Chen, X and Wen, R and Ma, P and Gu, K and Li, C and Zhou, C and Lei, C and Tang, Y and Wang, H}, title = {Immunocapture Magnetic Beads Enhanced the LAMP-CRISPR/Cas12a Method for the Sensitive, Specific, and Visual Detection of Campylobacter jejuni.}, journal = {Biosensors}, volume = {12}, number = {3}, pages = {}, pmid = {35323424}, issn = {2079-6374}, support = {31830098//National Natural Science Foundation of China/ ; CARS-40-K14//National System for Layer Production Technology/ ; SCU2019D013//Fundamental Research Funds for the Central Universities/ ; 2020NZZJ001//Sichuan Science and Technology Program/ ; 2021ZDZX0010//Sichuan Science and Technology Program/ ; 2019YFH0077//International Cooperation Program in Sichuan Province/ ; }, mesh = {CRISPR-Cas Systems ; *Campylobacter jejuni ; Magnetic Phenomena ; Molecular Diagnostic Techniques ; Nucleic Acid Amplification Techniques/methods ; }, abstract = {Campylobacter jejuni is one of the most important causes of food-borne infectious disease, and poses challenges to food safety and public health. Establishing a rapid, accurate, sensitive, and simple detection method for C. jejuni enables early diagnosis, early intervention, and prevention of pathogen transmission. In this study, an immunocapture magnetic bead (ICB)-enhanced loop-mediated isothermal amplification (LAMP) CRISPR/Cas12a method (ICB-LAMP-CRISPR/Cas12a) was developed for the rapid and visual detection of C. jejuni. Using the ICB-LAMP-CRISPR/Cas12a method, C. jejuni was first captured by ICB, and the bacterial genomic DNA was then released by heating and used in the LAMP reaction. After the LAMP reaction, LAMP products were mixed and detected by the CRISPR/Cas12a cleavage mixture. This ICB-LAMP-CRISPR/Cas12a method could detect a minimum of 8 CFU/mL of C. jejuni within 70 min. Additionally, the method was performed in a closed tube in addition to ICB capture, which eliminates the need to separate preamplification and transfer of amplified products to avoid aerosol pollution. The ICB-LAMP-CRISPR/Cas12a method was further validated by testing 31 C. jejuni-positive fecal samples from different layer farms. This method is an all-in-one, simple, rapid, ultrasensitive, ultraspecific, visual detection method for instrument-free diagnosis of C. jejuni, and has wide application potential in future work.}, }
@article {pmid35322386, year = {2022}, author = {Bykonya, AG and Lavrov, AV and Smirnikhina, SA}, title = {Methods for CRISPR-Cas as Ribonucleoprotein Complex Delivery In Vivo.}, journal = {Molecular biotechnology}, volume = {}, number = {}, pages = {}, pmid = {35322386}, issn = {1559-0305}, abstract = {The efficient delivery of CRISPR-Cas components is still a key and unsolved problem. CRISPR-Cas delivery in the form of a Cas protein+sgRNA (ribonucleoprotein complex, RNP complex), has proven to be extremely effective, since it allows to increase on-target activity, while reducing nonspecific activity. The key point for in vivo genome editing is the direct delivery of artificial nucleases and donor DNA molecules into the somatic cells of an adult organism. At the same time, control of the dose of artificial nucleases is impossible, which affects the efficiency of genome editing in the affected cells. Poor delivery efficiency and low editing efficacy reduce the overall potency of the in vivo genome editing process. Here we review how this problem is currently being solved in scientific works and what types of in vivo delivery methods of Cas9/sgRNA RNPs have been developed.}, }
@article {pmid35322099, year = {2022}, author = {Heu, CC and Gross, RJ and Le, KP and LeRoy, DM and Fan, B and Hull, JJ and Brent, CS and Fabrick, JA}, title = {CRISPR-mediated knockout of cardinal and cinnabar eye pigmentation genes in the western tarnished plant bug.}, journal = {Scientific reports}, volume = {12}, number = {1}, pages = {4917}, pmid = {35322099}, issn = {2045-2322}, mesh = {Animals ; CRISPR-Cas Systems ; *Eye Color/genetics ; Gene Editing ; *Heteroptera/genetics ; Mercury Compounds ; Nymph ; Pigmentation/genetics ; Plants/genetics ; }, abstract = {The western tarnished plant bug, Lygus hesperus, is a key hemipteran pest of numerous agricultural, horticultural, and industrial crops in the western United States and Mexico. A lack of genetic tools in L. hesperus hinders progress in functional genomics and in developing innovative pest control methods such as gene drive. Here, using RNA interference (RNAi) against cardinal (LhCd), cinnabar (LhCn), and white (LhW), we showed that knockdown of LhW was lethal to developing embryos, while knockdown of LhCd or LhCn produced bright red eye phenotypes, in contrast to wild-type brown eyes. We further used CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) genome editing to generate germline knockouts of both LhCd (Card) and LhCn (Cinn), producing separate strains of L. hesperus characterized by mutant eye phenotypes. Although the cardinal knockout strain Card exhibited a gradual darkening of the eyes to brown typical of the wild-type line later in nymphal development, we observed bright red eyes throughout all life stages in the cinnabar knockout strain Cinn, making it a viable marker for tracking gene editing in L. hesperus. These results provide evidence that CRISPR/Cas9 gene editing functions in L. hesperus and that eye pigmentation genes are useful for tracking the successful genetic manipulation of this insect.}, }
@article {pmid35320824, year = {2022}, author = {Monte Neto, RLD and Moreira, POL and de Sousa, AM and Garcia, MADN and Maran, SR and Moretti, NS}, title = {Antileishmanial metallodrugs and the elucidation of new drug targets linked to post-translational modifications machinery: pitfalls and progress.}, journal = {Memorias do Instituto Oswaldo Cruz}, volume = {117}, number = {}, pages = {e210403}, pmid = {35320824}, issn = {1678-8060}, mesh = {*Antiprotozoal Agents/chemistry ; Drug Discovery ; Humans ; *Leishmania ; *Leishmaniasis/drug therapy ; Protein Processing, Post-Translational ; }, abstract = {Despite the increasing number of manuscripts describing potential alternative antileishmanial compounds, little is advancing on translating these knowledges to new products to treat leishmaniasis. This is in part due to the lack of standardisations during pre-clinical drug discovery stage and also depends on the alignment of goals among universities/research centers, government and pharmaceutical industry. Inspired or not by drug repurposing, metal-based antileishmanial drugs represent a class that deserves more attention on its use for leishmaniasis chemotherapy. Together with new chemical entities, progresses have been made on the knowledge of parasite-specific drug targets specially after using CRISPR/Cas system for functional studies. In this regard, Leishmania parasites undergoe post-translational modification as key regulators in several cellular processes, which represents an entire new field for drug target elucidation, once this is poorly explored. This perspective review describes the advances on antileishmanial metallodrugs and the elucidation of drug targets based on post-translational modifications, highlighting the limitations on the drug discovery/development process and suggesting standardisations focused on products addressed to who need it most.}, }
@article {pmid35320636, year = {2022}, author = {Alekseeva, AE and Brusnigina, NF and Gordinskaya, NA and Makhova, MA and Kolesnikova, EA}, title = {Molecular genetic characteristics of resistome and virulome of carbapenem-resistant Klebsiella pneumoniae clinical strains.}, journal = {Klinicheskaia laboratornaia diagnostika}, volume = {67}, number = {3}, pages = {186-192}, doi = {10.51620/0869-2084-2022-67-3-186-192}, pmid = {35320636}, issn = {0869-2084}, mesh = {Anti-Bacterial Agents/pharmacology ; Carbapenems/pharmacology ; Humans ; *Klebsiella Infections/drug therapy/genetics ; *Klebsiella pneumoniae/genetics ; Microbial Sensitivity Tests ; Molecular Biology ; }, abstract = {The characteristics of resistome and virulome structure of four carbapenem-resistant Klebsiella pneumoniae clinical strains are present in the work. Two strains belonged to the sequence-type ST395, one strain - ST2262, one strain - to the new sequence-type 5816. The genes of fimbriae, enterobactin, beta-lactamase SHV type, resistance to fosfomycin fosA and transport of fluoroquinolones oqxAB in all Klebsiella strains chromosome structure were identified. The determinants of yersineobactin and aerobactin are enriched the virulome of ST395 NNKP315 and NNKP343 strains. The aerobactin genes are located on IncHI1B plasmids (IncHI1B/FIB) which highly homologous to the virulence pLVPK and pK2044 plasmids. IncR, IncL, IncQ plasmids carrying blaOXA-48, blaCTX-M-15, blaOXA-1, blaTEM-1, qnrS1, tetA, sul1, dfrA1, aac(6 ')-Ib-cr, catA1, catB3 etc. were identified in these strains. As a result of in silico analysis, an assumption about the localization of the blaOXA-48 in the structure of the IncHI1B plasmid of NNKP315 strain was made. This plasmid also contains the aminoglycosidases genes inserted into a class 1 integron In822. The mutations were found in the porin proteins OmpK35, OmpK36 and OmpK37 genes, which increases the carbapenem resistance. The virulome of NNKP16 (ST2262) strain additionally includes of the iron utilization system kfuABC chromosomal genes, and the virulome of NNKP15 (ST5816) strain contains of the capsular polysaccharide kvgAS and microcin E492 genes. Additional determinants of resistance were not identified in the resistome structure of K. pneumoniae NNKP16 and only the blaCTX-M-15 gene was found in the NNKP15 strain. The absence of acquired resistance genes seems to be due to the presence of the type I-E CRISPR-Cas system. Multiple drug resistance of the studied strains is associated with mutations identified in the gene structure of porin proteins OmpK36 and OmpK37, as well as the activity of efflux systems. It was showed the stop codon formation in the nucleotide sequence of the regulatory gene ramR to both strains, which can potentially provide overexpression of AcrAB efflux proteins.}, }
@article {pmid35320567, year = {2022}, author = {Martin, CJ and Calarco, JA}, title = {Approaches for CRISPR/Cas9 Genome Editing in C. elegans.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2468}, number = {}, pages = {215-237}, pmid = {35320567}, issn = {1940-6029}, mesh = {Animals ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; *Caenorhabditis elegans/genetics ; *Gene Editing/methods ; *Genome, Helminth ; }, abstract = {The clustered, regularly interspaced, short, palindromic repeat (CRISPR)-associated (CAS) nuclease Cas9 has been used in many organisms to generate specific mutations and transgene insertions. Here we describe our most up-to-date protocols using the S. pyogenes Cas9 in C. elegans that provides a convenient and effective approach for making heritable changes to the worm genome. We present several considerations when deciding which strategy best suits the needs of the experiment.}, }
@article {pmid35320539, year = {2022}, author = {Feng, C and Nita-Lazar, M and González-Montalbán, N and Wang, J and Mancini, J and Wang, S and Ravindran, C and Ahmed, H and Vasta, GR}, title = {Manipulating Galectin Expression in Zebrafish (Danio rerio).}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2442}, number = {}, pages = {425-443}, pmid = {35320539}, issn = {1940-6029}, mesh = {Animals ; *Galectins/metabolism ; Gene Knockdown Techniques ; Mammals/genetics ; Morpholinos/genetics/metabolism ; RNA/metabolism ; *Zebrafish/metabolism ; }, abstract = {Techniques for disrupting gene expression are invaluable tools for the analysis of the biological role of a gene product. Because of its genetic tractability and multiple advantages over conventional mammalian models, the zebrafish (Danio rerio) is recognized as a powerful system for gaining new insight into diverse aspects of human health and disease. Among the multiple mammalian gene families for which the zebrafish has shown promise as an invaluable model for functional studies, the galectins have attracted great interest due to their participation in early development, regulation of immune homeostasis, and recognition of microbial pathogens. Galectins are β-galactosyl-binding lectins with a characteristic sequence motif in their carbohydrate recognition domains (CRDs), that constitute an evolutionary conserved family ubiquitous in eukaryotic taxa. Galectins are emerging as key players in the modulation of many important pathological processes, which include acute and chronic inflammatory diseases, autoimmunity and cancer, thus making them potential molecular targets for innovative drug discovery. Here, we provide a review of the current methods available for the manipulation of gene expression in the zebrafish, with a focus on gene knockdown [morpholino (MO)-derived antisense oligonucleotides] and knockout (CRISPR-Cas) technologies.}, }
@article {pmid35320319, year = {2022}, author = {Snyder, AJ and Abad, AT and Danthi, P}, title = {A CRISPR-Cas9 screen reveals a role for WD repeat-containing protein 81 (WDR81) in the entry of late penetrating viruses.}, journal = {PLoS pathogens}, volume = {18}, number = {3}, pages = {e1010398}, pmid = {35320319}, issn = {1553-7374}, support = {R01 AI110637/AI/NIAID NIH HHS/United States ; R03 AI142013/AI/NIAID NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems ; Endosomes/metabolism ; Mammals ; Mice ; *Reoviridae/genetics ; *Reoviridae Infections/metabolism ; WD40 Repeats ; }, abstract = {Successful initiation of infection by many different viruses requires their uptake into the endosomal compartment. While some viruses exit this compartment early, others must reach the degradative, acidic environment of the late endosome. Mammalian orthoreovirus (reovirus) is one such late penetrating virus. To identify host factors that are important for reovirus infection, we performed a CRISPR-Cas9 knockout (KO) screen that targets over 20,000 genes in fibroblasts derived from the embryos of C57/BL6 mice. We identified seven genes (WDR81, WDR91, RAB7, CCZ1, CTSL, GNPTAB, and SLC35A1) that were required for the induction of cell death by reovirus. Notably, CRISPR-mediated KO of WD repeat-containing protein 81 (WDR81) rendered cells resistant to reovirus infection. Susceptibility to reovirus infection was restored by complementing KO cells with human WDR81. Although the absence of WDR81 did not affect viral attachment efficiency or uptake into the endosomal compartments for initial disassembly, it reduced viral gene expression and diminished infectious virus production. Consistent with the role of WDR81 in impacting the maturation of endosomes, WDR81-deficiency led to the accumulation of reovirus particles in dead-end compartments. Though WDR81 was dispensable for infection by VSV (vesicular stomatitis virus), which exits the endosomal system at an early stage, it was required for VSV-EBO GP (VSV that expresses the Ebolavirus glycoprotein), which must reach the late endosome to initiate infection. These results reveal a previously unappreciated role for WDR81 in promoting the replication of viruses that transit through late endosomes.}, }
@article {pmid35318087, year = {2022}, author = {Jiang, J and Sun, Y and Wang, Y and Sabek, A and Shangguan, A and Wang, K and Zhao, S and Li, G and Zhou, A and Zhang, S}, title = {Genome-wide CRISPR/Cas9 screen identifies host factors important for porcine reproductive and respiratory syndrome virus replication.}, journal = {Virus research}, volume = {314}, number = {}, pages = {198738}, doi = {10.1016/j.virusres.2022.198738}, pmid = {35318087}, issn = {1872-7492}, mesh = {Animals ; CRISPR-Cas Systems ; Genome ; *Porcine Reproductive and Respiratory Syndrome/genetics ; *Porcine respiratory and reproductive syndrome virus/genetics ; Receptors, Cell Surface/genetics ; Swine ; Virus Replication ; }, abstract = {Porcine reproductive and respiratory syndrome (PRRS), a viral infection caused by PRRS virus (PRRSV) can result in severe reproductive failure, and respiratory disease in the pigs thus causing enormous economic losses to the global swine industry. Although the cellular receptors for PRRSV have been identified, but mechanisms underlying PPRSV replication remain obscure. Here, we have performed a genome-scale CRISPR/Cas9 knockout screen in the pig kidney cells with PRRSV. Several genes were found to be highly enriched post-PRRSV selection, just like KxDL Motif Containing 1(KXD1), Proteasome 26S Subunit, Non-ATPase 3 (PSMD3) and Galectin 2 (LGALS2) and soon on. Importantly, we have identified that loss of KXD1 resulted in the restricted autophagy and inhibited replication of PRRSV. Therefore, our study demonstrates that CRISPR/Cas9 system can be effectively used for the screening of pig factors responsible for PRRSV replication.}, }
@article {pmid35316854, year = {2022}, author = {Bonillo, M and Pfromm, J and Fischer, MD}, title = {Challenges to Gene Editing Approaches in the Retina.}, journal = {Klinische Monatsblatter fur Augenheilkunde}, volume = {239}, number = {3}, pages = {275-283}, doi = {10.1055/a-1757-9810}, pmid = {35316854}, issn = {1439-3999}, mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genetic Therapy/methods ; Humans ; Retina ; *Retinal Diseases/genetics ; }, abstract = {Retinal gene therapy has recently been at the cutting edge of clinical development in the diverse field of genetic therapies. The retina is an attractive target for genetic therapies such as gene editing due to the distinctive anatomical and immunological features of the eye, known as immune privilege, so that inherited retinal diseases (IRDs) have been studied in several clinical studies. Thus, rapid strides are being made toward developing targeted treatments for IRDs. Gene editing in the retina faces a group of heterogenous challenges, including editing efficiencies, off-target effects, the anatomy of the target organ, immune responses, inactivation, and identifying optimal application methods. As clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) based technologies are at the forefront of current gene editing advances, their specific editing efficiency challenges and potential off-target effects were assessed. The immune privilege of the eye reduces the likelihood of systemic immune responses following retinal gene therapy, but possible immune responses must not be discounted. Immune responses to gene editing in the retina may be humoral or cell mediated, with immunologically active cells, including microglia, implicated in facilitating possible immune responses to gene editing. Immunogenicity of gene therapeutics may also lead to the inactivation of edited cells, reducing potential therapeutic benefits. This review outlines the broad spectrum of potential challenges currently facing retinal gene editing, with the goal of facilitating further advances in the safety and efficacy of gene editing therapies.}, }
@article {pmid35316773, year = {2022}, author = {Mir, TUG and Wani, AK and Akhtar, N and Shukla, S}, title = {CRISPR/Cas9: Regulations and challenges for law enforcement to combat its dual-use.}, journal = {Forensic science international}, volume = {334}, number = {}, pages = {111274}, doi = {10.1016/j.forsciint.2022.111274}, pmid = {35316773}, issn = {1872-6283}, mesh = {*CRISPR-Cas Systems ; DNA/genetics ; Gene Editing ; Humans ; *Law Enforcement ; Plants ; }, abstract = {For many years, scientists have aspired to edit any DNA segment of living organisms precisely, and with the discovery of CRISPR/Cas9, they have moved closer to achieving their objective. Using this tool, researchers can knock out or knock-in a gene into or from any part of the genome. The CRISPR-Cas9 system is a highly effective and accurate tool and has replaced previous genome editing technologies such as ZFN and TALEN having lesser precision and accuracy. Advances in CRISPR/Cas technology have been remarkable in recent years, with significant applications in various fields of life sciences research. Despite the diverse applications of the CRISPR system, this tool also poses a threat to living organisms and the environment. The intentional misuse of CRISPR could be a disaster to society and the environment. Different countries have imposed laws and guidelines to regulate genome editing through multiple agencies. However, in some countries, these rules and regulations are still under development or don't exist, leading to higher chances of the technology getting misused. Dual-use of this technology could be a threat to biosecurity and biodefense. This article summarizes the potential applications of the CRISPR system in human and plants, and also evaluate the laws and regulations imposed by different countries to keep genome editing technology under check.}, }
@article {pmid35316566, year = {2022}, author = {Schiermeyer, A and Cerda-Bennasser, P and Schmelter, T and Huang, X and Christou, P and Schillberg, S}, title = {Rapid production of SaCas9 in plant-based cell-free lysate for activity testing.}, journal = {Biotechnology journal}, volume = {}, number = {}, pages = {e2100564}, doi = {10.1002/biot.202100564}, pmid = {35316566}, issn = {1860-7314}, abstract = {Cas9 nucleases have become the most versatile tool for genome editing projects in a broad range of organisms. The recombinant production of Cas9 nuclease is desirable for in vitro activity assays or the preparation of ribonucleoproteins (RNPs) for DNA-free genome editing approaches. For the rapid production of Cas9, we explored the use of a recently established cell-free lysate from tobacco (Nicotiana tabacum L.) BY-2 cells. Using this system, the 130-kDa Cas9 nuclease from Staphylococcus aureus (SaCas9) was produced and subsequently purified via affinity chromatography. The purified apoenzyme was supplemented with 10 different sgRNAs, and the nuclease activity was confirmed by the linearization of plasmid DNA containing cloned DNA target sequences.}, }
@article {pmid35315725, year = {2022}, author = {Ye, Y and Shi, Q and Yang, T and Xie, F and Zhang, X and Xu, B and Fang, J and Chen, J and Zhang, Y and Li, J}, title = {In Vivo Visualized Tracking of Tumor-Derived Extracellular Vesicles Using CRISPR-Cas9 System.}, journal = {Technology in cancer research &amp; treatment}, volume = {21}, number = {}, pages = {15330338221085370}, pmid = {35315725}, issn = {1533-0338}, mesh = {CRISPR-Cas Systems/genetics ; Cell Communication ; *Extracellular Vesicles/genetics/metabolism ; Gene Editing ; Humans ; *Melanoma/genetics/metabolism ; }, abstract = {Introduction: Tumor extracellular vesicles (EVs) and their relevance to various processes of tumor growth have been vigorously investigated over the past decade. However, obtaining direct evidence of spontaneous EV transfer in vivo remains challenging. In our previous study, a single-guide RNA (sgRNA): Cas9 ribonucleoprotein complex, which can efficiently delete target genes, was delivered into recipient cells using an engineered EV. Aim: Applying this newly discovered exosomal bio-cargo to track the uptake and distribution of tumor EVs. Methods: Tumor cells of interest were engineered to express and release the sgRNA:Cas9 complex, and a reporter cell/system containing STOP-fluorescent protein (FP) elements was also generated. EV-delivered Cas9 proteins from donor cells were programmed by a pair of sgRNAs to completely delete a blockade sequence and, in turn, recuperated the expression of FP in recipient reporter cells. Thus, fluorescently illuminated cells indicate the uptake of EVs. To improve the efficiency and sensitivity of this tracking system in vivo, we optimized the sgRNA design, which could more efficiently trigger the expression of reporter proteins. Results: We demonstrated the EV-mediated crosstalk between tumor cells, and between tumor cells and normal cells in vitro. In vivo, we showed that intravenously administered EVs can be taken up by the liver. Moreover, we showed that EVs derived from melanoma xenografts in vivo preferentially target the brain and liver. This distribution resembles the manifestation of organotrophic metastasis of melanoma. Conclusion: This study provides an alternative tool to study the distribution and uptake of tumor EVs.}, }
@article {pmid35315229, year = {2022}, author = {Mukherjee, P and Patino, CA and Pathak, N and Lemaitre, V and Espinosa, HD}, title = {Deep Learning-Assisted Automated Single Cell Electroporation Platform for Effective Genetic Manipulation of Hard-to-Transfect Cells.}, journal = {Small (Weinheim an der Bergstrasse, Germany)}, volume = {18}, number = {20}, pages = {e2107795}, doi = {10.1002/smll.202107795}, pmid = {35315229}, issn = {1613-6829}, support = {//U.S. Department of Energy/ ; //Office of Science/ ; //Basic Energy Sciences/ ; }, abstract = {Genome engineering of cells using CRISPR/Cas systems has opened new avenues for pharmacological screening and investigating the molecular mechanisms of disease. A critical step in many such studies is the intracellular delivery of the gene editing machinery and the subsequent manipulation of cells. However, these workflows often involve processes such as bulk electroporation for intracellular delivery and fluorescence activated cell sorting for cell isolation that can be harsh to sensitive cell types such as human-induced pluripotent stem cells (hiPSCs). This often leads to poor viability and low overall efficacy, requiring the use of large starting samples. In this work, a fully automated version of the nanofountain probe electroporation (NFP-E) system, a nanopipette-based single-cell electroporation method is presented that provides superior cell viability and efficiency compared to traditional methods. The automated system utilizes a deep convolutional network to identify cell locations and a cell-nanopipette contact algorithm to position the nanopipette over each cell for the application of electroporation pulses. The automated NFP-E is combined with microconfinement arrays for cell isolation to demonstrate a workflow that can be used for CRISPR/Cas9 gene editing and cell tracking with potential applications in screening studies and isogenic cell line generation.}, }
@article {pmid35314803, year = {2022}, author = {Lu, S and Tong, X and Han, Y and Zhang, K and Zhang, Y and Chen, Q and Duan, J and Lei, X and Huang, M and Qiu, Y and Zhang, DY and Zhou, X and Zhang, Y and Yin, H}, title = {Fast and sensitive detection of SARS-CoV-2 RNA using suboptimal protospacer adjacent motifs for Cas12a.}, journal = {Nature biomedical engineering}, volume = {6}, number = {3}, pages = {286-297}, pmid = {35314803}, issn = {2157-846X}, mesh = {*COVID-19/diagnosis ; CRISPR-Cas Systems ; Humans ; *RNA, Viral/genetics ; SARS-CoV-2/genetics ; Sensitivity and Specificity ; }, abstract = {CRISPR-based assays for the detection of nucleic acids are highly specific, yet they are not fast, sensitive or easy to use. Here we report a one-step fluorescence assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in nasopharyngeal samples, with a sample-to-answer time of less than 20 minutes and a sensitivity comparable to that of quantitative real-time PCR with reverse transcription (RT-qPCR). The assay uses suboptimal protospacer adjacent motifs, allowing for flexibility in the design of CRISPR RNAs and slowing down the kinetics of Cas12a-mediated collateral cleavage of fluorescent DNA reporters and cis cleavage of substrates, which leads to stronger fluorescence owing to the accumulation of amplicons generated by isothermal recombinase polymerase amplification. In a set of 204 nasopharyngeal samples with RT-qPCR cycle thresholds ranging from 18.1 to 35.8, the assay detected SARS-CoV-2 with a sensitivity of 94.2% and a specificity of 100%, without the need for RNA extraction. Rapid and sensitive assays for nucleic acid testing in one pot that allow for flexibility in assay design may aid the development of reliable point-of-care nucleic acid testing.}, }
@article {pmid35314780, year = {2022}, author = {Liao, C and Sharma, S and Svensson, SL and Kibe, A and Weinberg, Z and Alkhnbashi, OS and Bischler, T and Backofen, R and Caliskan, N and Sharma, CM and Beisel, CL}, title = {Spacer prioritization in CRISPR-Cas9 immunity is enabled by the leader RNA.}, journal = {Nature microbiology}, volume = {7}, number = {4}, pages = {530-541}, pmid = {35314780}, issn = {2058-5276}, support = {BE 6703/1-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; SH 580/9-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; BA 2168/23-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 865973//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; 865973/ERC_/European Research Council/International ; Z-6//Interdisziplinäres Zentrum für Klinische Forschung, Universitätsklinikum Würzburg (Interdisciplinary Center for Clinical Research, University Hospital of Würzburg)/ ; }, mesh = {*Bacteriophages/genetics/metabolism ; *CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems ; RNA/genetics ; Streptococcus pyogenes/genetics/metabolism ; }, abstract = {CRISPR-Cas systems store fragments of foreign DNA, called spacers, as immunological recordings used to combat future infections. Of the many spacers stored in a CRISPR array, the most recent are known to be prioritized for immune defence. However, the underlying mechanism remains unclear. Here we show that the leader region upstream of CRISPR arrays in CRISPR-Cas9 systems enhances CRISPR RNA (crRNA) processing from the newest spacer, prioritizing defence against the matching invader. Using the CRISPR-Cas9 system from Streptococcus pyogenes as a model, we found that the transcribed leader interacts with the conserved repeats bordering the newest spacer. The resulting interaction promotes transactivating crRNA (tracrRNA) hybridization with the second of the two repeats, accelerating crRNA processing. Accordingly, disruption of this structure reduces the abundance of the associated crRNA and immune defence against targeted plasmids and bacteriophages. Beyond the S. pyogenes system, bioinformatics analyses revealed that leader-repeat structures appear across CRISPR-Cas9 systems. CRISPR-Cas systems thus possess an RNA-based mechanism to prioritize defence against the most recently encountered invaders.}, }
@article {pmid35314679, year = {2022}, author = {Schindele, A and Gehrke, F and Schmidt, C and Röhrig, S and Dorn, A and Puchta, H}, title = {Using CRISPR-Kill for organ specific cell elimination by cleavage of tandem repeats.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {1502}, pmid = {35314679}, issn = {2041-1723}, mesh = {*Arabidopsis/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Endonucleases/metabolism ; Mutagenesis ; Tandem Repeat Sequences ; }, abstract = {CRISPR/Cas has been mainly used for mutagenesis through the induction of double strand breaks (DSBs) within unique protein-coding genes. Using the SaCas9 nuclease to induce multiple DSBs in functional repetitive DNA of Arabidopsis thaliana, we can now show that cell death can be induced in a controlled way. This approach, named CRISPR-Kill, can be used as tool for tissue engineering. By simply exchanging the constitutive promoter of SaCas9 with cell type-specific promoters, it is possible to block organogenesis in Arabidopsis. By AP1-specific expression of CRISPR-Kill, we are able to restore the apetala1 phenotype and to specifically eliminate petals. In addition, by expressing CRISPR-Kill in root-specific pericycle cells, we are able to dramatically reduce the number and the length of lateral roots. In the future, the application of CRISPR-Kill may not only help to control development but could also be used to change the biochemical properties of plants.}, }
@article {pmid35314527, year = {2022}, author = {Jair Lara-Navarro, I and Rebeca Jaloma-Cruz, A}, title = {Current Therapies in Hemophilia: From Plasma-Derived Factor Modalities to CRISPR/Cas Alternatives.}, journal = {The Tohoku journal of experimental medicine}, volume = {256}, number = {3}, pages = {197-207}, doi = {10.1620/tjem.256.197}, pmid = {35314527}, issn = {1349-3329}, mesh = {CRISPR-Cas Systems/genetics ; Factor IX/genetics/therapeutic use ; *Hemophilia A/genetics/therapy ; *Hemophilia B/drug therapy/therapy ; Humans ; Quality of Life ; }, abstract = {Since the middle of the last century, there have been amazing therapeutic advances for hemophilia such as the development of plasma-derived products and bioengineered recombinant factors VIII and IX (for hemophilia A and B, respectively) with improved stability, higher activity, and extended half-life. The recent use of a monoclonal antibody that mimics factor VIII activity (which is an efficient treatment for all hemophilia A phenotypes with or without inhibitors) has shown the great possibilities of non-factor therapies for improving the quality of life of hemophilia A patients, with a safer application and long-lasting effects. Gene therapy offers the promise of a "true cure" for hemophilia based on the permanent effect that a gene edition may render. Clinical trials developed in the last decade based on adenoviral vectors show modest but consistent results; now, CRISPR/Cas technology (which is considered the most efficient tool for gene edition) is being developed on different hemophilia models. Once the off-target risks are solved and an efficient switch on/off for Cas activity is developed, this strategy might become the most feasible option for gene therapy in hemophilia and other monogenic diseases.}, }
@article {pmid35313109, year = {2022}, author = {Harrison, PT}, title = {CRISPR gene editing - what are the possibilities for respiratory medicine?.}, journal = {Expert review of respiratory medicine}, volume = {16}, number = {4}, pages = {371-374}, doi = {10.1080/17476348.2022.2056021}, pmid = {35313109}, issn = {1747-6356}, mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genetic Therapy ; Humans ; *Pulmonary Medicine ; }, }
@article {pmid35312212, year = {2022}, author = {Nguyen, STT and Vardeh, DP and Nelson, TM and Pearson, LA and Kinsela, AS and Neilan, BA}, title = {Bacterial community structure and metabolic potential in microbialite-forming mats from South Australian saline lakes.}, journal = {Geobiology}, volume = {}, number = {}, pages = {}, doi = {10.1111/gbi.12489}, pmid = {35312212}, issn = {1472-4669}, support = {DP1093106//Australian Research Council/ ; FF0883440//Australian Research Council/ ; }, abstract = {Microbialites are sedimentary rocks created in association with benthic microorganisms. While they harbour complex microbial communities, Cyanobacteria perform critical roles in sediment stabilisation and accretion. Microbialites have been described from permanent and ephemeral saline lakes in South Australia; however, the microbial communities that generate and inhabit these biogeological structures have not been studied in detail. To address this knowledge gap, we investigated the composition, diversity and metabolic potential of bacterial communities from different microbialite-forming mats and surrounding sediments in five South Australian saline coastal lakes using 16S rRNA gene sequencing and predictive metagenome analyses. While Proteobacteria and Bacteroidetes were the dominant phyla recovered from the mats and sediments, Cyanobacteria were significantly more abundant in the mat samples. Interestingly, at lower taxonomic levels, the mat communities were vastly different across the five lakes. Comparative analysis of putative mat and sediment metagenomes via PICRUSt2 revealed important metabolic pathways driving the process of carbonate precipitation, including cyanobacterial oxygenic photosynthesis, ureolysis and nitrogen fixation. These pathways were highly conserved across the five examined lakes, although they appeared to be performed by distinct groups of bacterial taxa found in each lake. Stress response, quorum sensing and circadian clock were other important pathways predicted by the in silico metagenome analysis. The enrichment of CRISPR/Cas and phage shock associated genes in these cyanobacteria-rich communities suggests that they may be under selective pressure from viral infection. Together, these results highlight that a very stable ecosystem function is maintained by distinctly different communities in microbialite-forming mats in the five South Australian lakes and reinforce the concept that 'who' is in the community is not as critical as their net metabolic capacity.}, }
@article {pmid35310854, year = {2022}, author = {Liu, X and Qiu, X and Xu, S and Che, Y and Han, L and Kang, Y and Yue, Y and Chen, S and Li, F and Li, Z}, title = {A CRISPR-Cas12a-Assisted Fluorescence Platform for Rapid and Accurate Detection of Nocardia cyriacigeorgica.}, journal = {Frontiers in cellular and infection microbiology}, volume = {12}, number = {}, pages = {835213}, pmid = {35310854}, issn = {2235-2988}, mesh = {*CRISPR-Cas Systems ; DNA, Single-Stranded ; *Nocardia/genetics ; Nucleic Acid Amplification Techniques/methods ; }, abstract = {Nocardia cyriacigeorgica has gradually become a common pathogen in clinical microbial infections. Identification of Nocardia at the species level is essential to assess the susceptibility and pathogenicity of antimicrobials. However, there is no suitable method for rapid and accurate laboratory detection of N. cyriacigeorgica. In this study, we combined PCR amplification with the CRISPR-Cas12a system to establish a novel detection platform, named CRISPR-PCR, and applied it to the detection of N. cyriacigeorgica in clinical samples. The Cas12a protein exhibited collateral cleavage activity following CRISPR RNA binding to specific targets, then indiscriminately cleaved nearby single-stranded DNA, and this was evaluated for diagnostic nucleic acid detection by measuring the fluorescence signal using a fluorescence reader. The assay takes only 2 h, including DNA extraction for 20 min, nucleic acid pre-amplification for 70 min, and fluorescence detection for 20 min. The limit of detection for N. cyriacigeorgica was 10-3 ng and the specificity was 100%. Thus, the N. cyriacigeorgica CRISPR-PCR assay is a rapid and specific method for detecting N. cyriacigeorgica, and the CRISPR-PCR fluorescence detection platform has great potential for detection of other pathogens.}, }
@article {pmid35310559, year = {2022}, author = {Zhang, Y and Li, Z and Milon Essola, J and Ge, K and Dai, X and He, H and Xiao, H and Weng, Y and Huang, Y}, title = {Biosafety materials: Ushering in a new era of infectious disease diagnosis and treatment with the CRISPR/Cas system.}, journal = {Biosafety and health}, volume = {4}, number = {2}, pages = {70-78}, pmid = {35310559}, issn = {2590-0536}, abstract = {Despite multiple virus outbreaks over the past decade, including the devastating coronavirus disease 2019 (COVID-19) pandemic, the lack of accurate and timely diagnosis and treatment technologies has wreaked havoc on global biosecurity. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has the potential to address these critical needs for tackling infectious diseases to detect viral nucleic acids and inhibit viral replication. This review summarizes how the CRISPR/Cas system is being utilized for the treatment and diagnosis of infectious diseases with the help of biosafety materials and highlights the design principle and in vivo and in vitro efficacy of advanced biosafety materials used to deal with virus attacks.}, }
@article {pmid35308851, year = {2022}, author = {Chen, S and Wang, R and Peng, S and Xie, S and Lei, C and Huang, Y and Nie, Z}, title = {PAM-less conditional DNA substrates leverage trans-cleavage of CRISPR-Cas12a for versatile live-cell biosensing.}, journal = {Chemical science}, volume = {13}, number = {7}, pages = {2011-2020}, pmid = {35308851}, issn = {2041-6520}, abstract = {The CRISPR-Cas system has been repurposed as a powerful live-cell imaging tool, but its utility is limited to genomic loci and mRNA imaging in living cells. Here, we demonstrated the potential of the CRISPR-Cas system as a generalizable live-cell biosensing tool by extending its applicability to monitor diverse intracellular biomolecules. In this work, we engineered a CRISPR-Cas12a system with a generalized stimulus-responsive switch mechanism based on PAM-less conditional DNA substrates (pcDNAs). The pcDNAs with stimulus-responsiveness toward a trigger were constructed from the DNA substrates featuring no requirement of a protospacer-adjacent motif (PAM) and a bubble structure. With further leveraging the trans-cleavage activity of CRISPR-Cas12a for signal reporting, we established a versatile CRISPR-based live-cell biosensing system. This system enabled the sensitive sensing of various intracellular biomolecules, such as telomerase, ATP, and microRNA-21, making it a helpful tool for basic biochemical research and disease diagnostics.}, }
@article {pmid35308373, year = {2022}, author = {Duan, G and Kan, B and Li, D and Song, H}, title = {Editorial: The CRISPR/Cas System in Pathogen Resistance, Virulence, Diagnosis and Typing.}, journal = {Frontiers in microbiology}, volume = {13}, number = {}, pages = {832152}, pmid = {35308373}, issn = {1664-302X}, }
@article {pmid35308131, year = {2022}, author = {Lax, C and Navarro-Mendoza, MI and Pérez-Arques, C and Navarro, E and Nicolás, FE and Garre, V}, title = {Transformation and CRISPR-Cas9-mediated homologous recombination in the fungus Rhizopus microsporus.}, journal = {STAR protocols}, volume = {3}, number = {1}, pages = {101237}, pmid = {35308131}, issn = {2666-1667}, mesh = {*CRISPR-Cas Systems/genetics ; DNA ; Homologous Recombination ; *Mucormycosis/genetics ; Rhizopus/genetics ; }, abstract = {Here, we describe a reliable approach for targeted DNA integrations in the genome of R. microsporus, one of the main causal agents of mucormycosis. We provide a strategy for stable, targeted integration of DNA templates by homologous recombination (HR) based on the CRISPR-Cas9 technology. This strategy opens a wide range of possibilities for the genetic modification of R. microsporus and will be useful for the study of mucormycosis. For complete details on the use and execution of this protocol, please refer to Lax et al. (2021).}, }
@article {pmid35307599, year = {2022}, author = {Dong, JF and Feng, CJ and Wang, P and Li, RQ and Zou, QH}, title = {Comparative genomics analysis of Acinetobacter baumannii multi-drug resistant and drug sensitive strains in China.}, journal = {Microbial pathogenesis}, volume = {165}, number = {}, pages = {105492}, doi = {10.1016/j.micpath.2022.105492}, pmid = {35307599}, issn = {1096-1208}, mesh = {*Acinetobacter baumannii ; Anti-Bacterial Agents/pharmacology ; Drug Resistance, Multiple ; Drug Resistance, Multiple, Bacterial/genetics ; Genomics ; Microbial Sensitivity Tests ; Plasmids/genetics ; }, abstract = {The incidence of multidrug-resistant Acinetobacter baumannii has posed a major challenge for clinical treatment. There is still a significant gap in understanding the mechanism causing multi-drug resistance (MDR). In this study, the genomes of 10 drug sensitive and 10 multi-drug resistant A.baumannii strains isolated from a hospital in China were sequenced and compared. The antibiotic resistance genes, virulence factors were determined and CRIPSR-Cas system along with prophages were detected. The results showed that MDR strains are significantly different from the drug sensitive strains in the CARD entries, patterns of sequences matching up to plasmids, VFDB entries and CRISPR-Cas system. MDR strains contain unique CARD items related to antibiotic resistance which are absent in sensitive strains. Furthermore, sequences from genomes of MDR strains can match up with plasmids from more diversified bacteria genera compared to drug sensitive strains. MDR strains also contain a lower level of CRISPR genes and larger amount of prophages, along with higher levels of spacer sequences. These findings provide new experimental evidences for the study of the antibiotic resistance mechanism of A. baumannii.}, }
@article {pmid35306273, year = {2022}, author = {Chen, P and Wang, L and Qin, P and Yin, BC and Ye, BC}, title = {An RNA-based catalytic hairpin assembly circuit coupled with CRISPR-Cas12a for one-step detection of microRNAs.}, journal = {Biosensors &amp; bioelectronics}, volume = {207}, number = {}, pages = {114152}, doi = {10.1016/j.bios.2022.114152}, pmid = {35306273}, issn = {1873-4235}, mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; Endonucleases ; *MicroRNAs/genetics ; Nucleic Acid Amplification Techniques/methods ; }, abstract = {CRISPR-Cas nuclease-based nucleic acid detection has exhibited extraordinary value in the field of molecular diagnostics, but it usually involves two separate reaction steps of nucleic acid amplification and Cas-based endpoint detection, resulting in the use of multiple enzymes, inconvenient operation, and potential carry-over contamination. Here, we propose an RNA-based catalytic hairpin assembly (CHA) circuit coupled with CRISPR-Cas12a for one-step detection of microRNAs (miRNAs) at an isothermal condition. This method relies on the rational design of a spacer-blocking crRNA as a bridge between the two systems. The target miRNA can specifically trigger RNA-based CHA and induce a configurational change of the blocked crRNAs into precursor crRNAs (pre-crRNAs), which can be processed into mature crRNAs to function by leveraging the inherent RNase activities of Cas12a. In this way, the developed circuit achieves a femtomolar detection limit and shows an accurate detection of miRNA levels in different cell lines. Therefore, our method would provide a new paradigm to develop miRNA detection methods based on the CRISPR/Cas system.}, }
@article {pmid35306148, year = {2022}, author = {Sun, X and Wang, DO and Wang, J}, title = {Targeted manipulation of m6A RNA modification through CRISPR-Cas-based strategies.}, journal = {Methods (San Diego, Calif.)}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.ymeth.2022.03.006}, pmid = {35306148}, issn = {1095-9130}, abstract = {N6-methyladenosine (m6A) is a reversible and prevalent internal modification in RNAs and can be dynamically modulated by methyltransferase and demethylase. Targeted manipulation of m6A RNA modification is critical in studying the functions of specific m6A sites as well as developing molecular therapies through targeting m6A. The CRISPR-Cas systems including CRISPR-Cas9 and CRISPR-Cas13 have been widely used to edit and modify specific nucleotides on DNA and RNA through fusing effective proteins such as enzymes with Cas9/13. Through taking advantage of the m6A methyltransferase and demethylase, a series of CRISPR-Cas-based methods have also been developed to manipulate the m6A methylation at specific RNA sites. This review summarizes the latest CRISPR-Cas13 and Cas9 toolkits for m6A site-specific manipulation, including fundamental components, on-target efficiency, editing window, PAM/PFS requirement, and subcellularly localized targeting as well as potential limitations. We thus aim to provide an overview to assist researchers to choose an optimal tool to manipulate m6A for different purposes and also point out possible optimization strategies.}, }
@article {pmid35305903, year = {2022}, author = {Bekaert, B and Boel, A and Cosemans, G and De Witte, L and Menten, B and Heindryckx, B}, title = {CRISPR/Cas gene editing in the human germline.}, journal = {Seminars in cell &amp; developmental biology}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.semcdb.2022.03.012}, pmid = {35305903}, issn = {1096-3634}, abstract = {The ease and efficacy of CRISPR/Cas9 germline gene editing in animal models paved the way to human germline gene editing (HGGE), by which permanent changes can be introduced into the embryo. Distinct genes can be knocked out to examine their function during embryonic development. Alternatively, specific sequences can be introduced which can be applied to correct disease-causing mutations. To date, it has been shown that the success of HGGE is dependent on various experimental parameters and that various hurdles (i.e. loss-of-heterozygosity and mosaicism) need to be overcome before clinical applications should be considered. Due to the shortage of human germline material and the ethical constraints concerning HGGE, alternative models such as stem cells have been evaluated as well, in terms of their predictive value on the genetic outcome for HGGE approaches. This review will give an overview of the state of the art of HGGE in oocytes and embryos, and its accompanying challenges.}, }
@article {pmid35304449, year = {2022}, author = {Wang, Y and Zhang, G and Meng, Q and Huang, S and Guo, P and Leng, Q and Sun, L and Liu, G and Huang, X and Liu, J}, title = {Precise tumor immune rewiring via synthetic CRISPRa circuits gated by concurrent gain/loss of transcription factors.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {1454}, pmid = {35304449}, issn = {2041-1723}, mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Regulatory Networks ; Humans ; *Neoplasms/genetics/therapy ; *Transcription Factors/genetics ; }, abstract = {Reinvigoration of antitumor immunity has recently become the central theme for the development of cancer therapies. Nevertheless, the precise delivery of immunotherapeutic activities to the tumors remains challenging. Here, we explore a synthetic gene circuit-based strategy for specific tumor identification, and for subsequently engaging immune activation. By design, these circuits are assembled from two interactive modules, i.e., an oncogenic TF-driven CRISPRa effector, and a corresponding p53-inducible off-switch (NOT gate), which jointly execute an AND-NOT logic for accurate tumor targeting. In particular, two forms of the NOT gate are developed, via the use of an inhibitory sgRNA or an anti-CRISPR protein, with the second form showing a superior performance in gating CRISPRa by p53 loss. Functionally, the optimized AND-NOT logic circuit can empower a highly specific and effective tumor recognition/immune rewiring axis, leading to therapeutic effects in vivo. Taken together, our work presents an adaptable strategy for the development of precisely delivered immunotherapy.}, }
@article {pmid35303871, year = {2022}, author = {Dimitri, A and Herbst, F and Fraietta, JA}, title = {Engineering the next-generation of CAR T-cells with CRISPR-Cas9 gene editing.}, journal = {Molecular cancer}, volume = {21}, number = {1}, pages = {78}, pmid = {35303871}, issn = {1476-4598}, mesh = {CRISPR-Cas Systems ; Gene Editing ; Humans ; Immunotherapy, Adoptive ; *Neoplasms/drug therapy/therapy ; Receptors, Antigen, T-Cell/genetics ; *Receptors, Chimeric Antigen/metabolism ; T-Lymphocytes ; }, abstract = {Chimeric Antigen Receptor (CAR) T-cells represent a breakthrough in personalized cancer therapy. In this strategy, synthetic receptors comprised of antigen recognition, signaling, and costimulatory domains are used to reprogram T-cells to target tumor cells for destruction. Despite the success of this approach in refractory B-cell malignancies, optimal potency of CAR T-cell therapy for many other cancers, particularly solid tumors, has not been achieved. Factors such as T-cell exhaustion, lack of CAR T-cell persistence, cytokine-related toxicities, and bottlenecks in the manufacturing of autologous products have hampered the safety, effectiveness, and availability of this approach. With the ease and accessibility of CRISPR-Cas9-based gene editing, it is possible to address many of these limitations. Accordingly, current research efforts focus on precision engineering of CAR T-cells with conventional CRISPR-Cas9 systems or novel editors that can install desired genetic changes with or without introduction of a double-stranded break (DSB) into the genome. These tools and strategies can be directly applied to targeting negative regulators of T-cell function, directing therapeutic transgenes to specific genomic loci, and generating reproducibly safe and potent allogeneic universal CAR T-cell products for on-demand cancer immunotherapy. This review evaluates several of the ongoing and future directions of combining next-generation CRISPR-Cas9 gene editing with synthetic biology to optimize CAR T-cell therapy for future clinical trials toward the establishment of a new cancer treatment paradigm.}, }
@article {pmid35303554, year = {2022}, author = {Zhang, L and Jiang, H and Zhu, Z and Liu, J and Li, B}, title = {Integrating CRISPR/Cas within isothermal amplification for point-of-Care Assay of nucleic acid.}, journal = {Talanta}, volume = {243}, number = {}, pages = {123388}, doi = {10.1016/j.talanta.2022.123388}, pmid = {35303554}, issn = {1873-3573}, mesh = {*COVID-19/diagnosis ; CRISPR-Cas Systems/genetics ; Humans ; Nucleic Acid Amplification Techniques ; *Nucleic Acids ; Point-of-Care Systems ; }, abstract = {Nucleic acid detection technology is now widely used in scientific research and clinical testing, such as infectious and genetic diseases screening, molecular diagnosis of tumors and pharmacogenomic research, which is also an important part of in vitro diagnostics (IVD). However, with the increasing requirements of diagnosis and treatment, existing nucleic acid detection technologies are facing challenges in dealing with the current problems (especially since the outbreak of coronavirus disease in 2019 (Covid-19)). Recently, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (CRISPR/Cas)-based diagnostics have become a hot spot of attention. CRISPR/Cas has been developed as a molecular detection tool besides scientific research in biology and medicine fields, and some CRISPR-based products have already been translated. It is known as the "next-generation molecular diagnostic technology" because of its advantages such as easy design and accurate identification. CRISPR/Cas relies on pre-amplification of target sequences and subsequent detection of Cas proteins. Combining the CRISPR/Cas system with various isothermal nucleic acid amplification strategies can generate amplified detection signals, enrich low abundance molecular targets, improve the specificity and sensitivity of analysis, and develop point-of-care (POC) diagnostic techniques. In this review, we analyze the current status of CRISPR/Cas systems and isothermal amplification, report the advantages of combining the two and summarize the recent progress with the integration of both technologies with POC sensors in the nucleic acid field. In addition, the challenges and future prospects of CRISPR technology combined with isothermal amplification strategies in biosensing and clinical applications are discussed.}, }
@article {pmid35303481, year = {2022}, author = {Roth, MO and Li, H}, title = {"X" marks the spot: Mining the gold in CasX for gene editing.}, journal = {Molecular cell}, volume = {82}, number = {6}, pages = {1083-1085}, pmid = {35303481}, issn = {1097-4164}, support = {R01 GM099604/GM/NIGMS NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Gold ; Humans ; }, abstract = {In this issue of Molecular Cell, Tsuchida et al. (2022) present a successful structure-guided effort in improving genome-editing efficiencies of CRISPR-CasX from Deltaproteobacteria (DpbCasX) and Planctomycetes (PlmCasX). Engineered variants that stabilize the active conformational state improved the catalytic efficiency by ∼10-20 fold in vitro and mean-editing efficiency by ∼2-3 fold in human cells.}, }
@article {pmid35303047, year = {2022}, author = {Ceballos-Garzon, A and Roman, E and Pla, J and Pagniez, F and Amado, D and Alméciga-Díaz, CJ and Le Pape, P and Parra-Giraldo, CM}, title = {CRISPR-Cas9 approach confirms Calcineurin-responsive zinc finger 1 (Crz1) transcription factor as a promising therapeutic target in echinocandin-resistant Candida glabrata.}, journal = {PloS one}, volume = {17}, number = {3}, pages = {e0265777}, pmid = {35303047}, issn = {1932-6203}, mesh = {Animals ; Antifungal Agents/pharmacology/therapeutic use ; CRISPR-Cas Systems/genetics ; Calcineurin/metabolism ; *Candida glabrata/genetics/metabolism ; Drug Resistance, Fungal/genetics ; *Echinocandins/pharmacology/therapeutic use ; Humans ; Mice ; Microbial Sensitivity Tests ; Transcription Factors/genetics/metabolism ; Zinc/metabolism ; Zinc Fingers ; }, abstract = {Invasive fungal infections, which kill more than 1.6 million patients each year worldwide, are difficult to treat due to the limited number of antifungal drugs (azoles, echinocandins, and polyenes) and the emergence of antifungal resistance. The transcription factor Crz1, a key regulator of cellular stress responses and virulence, is an attractive therapeutic target because this protein is absent in human cells. Here, we used a CRISPR-Cas9 approach to generate isogenic crz1Δ strains in two clinical isolates of caspofungin-resistant C. glabrata to analyze the role of this transcription factor in susceptibility to echinocandins, stress tolerance, biofilm formation, and pathogenicity in both non-vertebrate (Galleria mellonella) and vertebrate (mice) models of candidiasis. In these clinical isolates, CRZ1 disruption restores the susceptibility to echinocandins in both in vitro and in vivo models, and affects their oxidative stress response, biofilm formation, cell size, and pathogenicity. These results strongly suggest that Crz1 inhibitors may play an important role in the development of novel therapeutic agents against fungal infections considering the emergence of antifungal resistance and the low number of available antifungal drugs.}, }
@article {pmid35302756, year = {2022}, author = {Lu, D and Foley, CA and Birla, SV and Hepperla, AJ and Simon, JM and James, LI and Hathaway, NA}, title = {Bioorthogonal Chemical Epigenetic Modifiers Enable Dose-Dependent CRISPR Targeted Gene Activation in Mammalian Cells.}, journal = {ACS synthetic biology}, volume = {11}, number = {4}, pages = {1397-1407}, pmid = {35302756}, issn = {2161-5063}, support = {R01 CA242305/CA/NCI NIH HHS/United States ; R01 GM118653/GM/NIGMS NIH HHS/United States ; R01 GM132299/GM/NIGMS NIH HHS/United States ; R61 DA047023/DA/NIDA NIH HHS/United States ; }, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Mammals/genetics ; Nuclear Proteins/genetics/metabolism ; Tacrolimus Binding Proteins/genetics/metabolism ; Transcription Factors/genetics ; Transcriptional Activation ; }, abstract = {CRISPR-Cas9 systems have been developed to regulate gene expression by using either fusions to epigenetic regulators or, more recently, through the use of chemically mediated strategies. These approaches have armed researchers with new tools to examine the function of proteins by intricately controlling expression levels of specific genes. Here we present a CRISPR-based chemical approach that uses a new chemical epigenetic modifier (CEM) to hone to a gene targeted with a catalytically inactive Cas9 (dCas9) bridged to an FK506-binding protein (FKBP) in mammalian cells. One arm of the bifunctional CEM recruits BRD4 to the target site, and the other arm is composed of a bumped ligand that binds to a mutant FKBP with a compensatory hole at F36V. This bump-and-hole strategy allows for activation of target genes in a dose-dependent and reversible fashion with increased specificity and high efficacy, providing a new synthetic biology approach to answer important mechanistic questions in the future.}, }
@article {pmid35302142, year = {2022}, author = {Li, D and Duan, C and Cheng, W and Gong, Y and Yao, Y and Wang, X and Wang, Z and Xiang, Y}, title = {A simple and rapid method to assay SARS-CoV-2 RNA based on a primer exchange reaction.}, journal = {Chemical communications (Cambridge, England)}, volume = {58}, number = {28}, pages = {4484-4487}, doi = {10.1039/d2cc00488g}, pmid = {35302142}, issn = {1364-548X}, mesh = {*COVID-19/diagnosis ; CRISPR-Cas Systems/genetics ; Humans ; Nucleic Acid Amplification Techniques/methods ; RNA, Viral/genetics ; *SARS-CoV-2/genetics ; }, abstract = {A simple method is proposed in this work for the detection of SARS-CoV-2 RNA based on a primer exchange reaction (PER). By ingeniously integrating the PER cascade and CRISPR/cas12a system, this method can achieve convenient detection of the target RNA in 40 min and distinguish a single-base mutation from the target sequence, demonstrating its superior analytical performance.}, }
@article {pmid35302126, year = {2022}, author = {Zhou, M and Yin, Y and Shi, Y and Huang, Z and Shi, Y and Chen, M and Ke, G and Zhang, XB}, title = {Spherical nucleic acid reporter-based cascade CRISPR/Cas12a amplifier for stable and sensitive biosensing of circulating tumor DNA.}, journal = {Chemical communications (Cambridge, England)}, volume = {58}, number = {28}, pages = {4508-4511}, doi = {10.1039/d2cc00960a}, pmid = {35302126}, issn = {1364-548X}, mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; *Circulating Tumor DNA/genetics ; Nucleic Acid Amplification Techniques ; *Nucleic Acids ; }, abstract = {Stable and sensitive ctDNA biosensing in complex biological fluid is highly important but still remains a challenge. Herein, we develop a spherical nucleic acid reporter-based cascade CRISPR/Cas12a amplifier with improved stability and sensitivity (5 orders of magnitude).}, }
@article {pmid35301482, year = {2022}, author = {Xie, Y and Zhang, L and Gao, Z and Yin, P and Wang, H and Li, H and Chen, Z and Zhang, Y and Yang, M and Feng, Y}, title = {AcrIF5 specifically targets DNA-bound CRISPR-Cas surveillance complex for inhibition.}, journal = {Nature chemical biology}, volume = {}, number = {}, pages = {}, pmid = {35301482}, issn = {1552-4469}, support = {32000901//National Natural Science Foundation of China (National Science Foundation of China)/ ; 31822012//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, abstract = {CRISPR-Cas systems are prokaryotic antiviral systems, and phages use anti-CRISPR proteins (Acrs) to inactivate these systems. Here we present structural and functional analyses of AcrIF5, exploring its unique anti-CRISPR mechanism. AcrIF5 shows binding specificity only for the target DNA-bound form of the crRNA-guided surveillance (Csy) complex, but not the apo Csy complex from the type I-F CRISPR-Cas system. We solved the structure of the Csy-dsDNA-AcrIF5 complex, revealing that the conformational changes of the Csy complex caused by dsDNA binding dictate the binding specificity for the Csy-dsDNA complex by AcrIF5. Mechanistically, five AcrIF5 molecules bind one Csy-dsDNA complex, which destabilizes the helical bundle domain of Cas8f, thus preventing subsequent Cas2/3 recruitment. AcrIF5 exists in symbiosis with AcrIF3, which blocks Cas2/3 recruitment. This attack on the recruitment event stands in contrast to the conventional mechanisms of blocking binding of target DNA. Overall, our study reveals an unprecedented mechanism of CRISPR-Cas inhibition by AcrIF5.}, }
@article {pmid35301428, year = {2022}, author = {Cui, YR and Wang, SJ and Ma, T and Yu, P and Chen, J and Guo, T and Meng, G and Jiang, B and Dong, J and Liu, J}, title = {KPT330 improves Cas9 precision genome- and base-editing by selectively regulating mRNA nuclear export.}, journal = {Communications biology}, volume = {5}, number = {1}, pages = {237}, pmid = {35301428}, issn = {2399-3642}, mesh = {Active Transport, Cell Nucleus ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Editing ; Humans ; RNA, Messenger/genetics/metabolism ; }, abstract = {CRISPR-based genome engineering tools are associated with off-target effects that constitutively active Cas9 protein may instigate. Previous studies have revealed the feasibility of modulating Cas9-based genome- and base-editing tools using protein or small-molecule CRISPR inhibitors. Here we screened a set of small molecule compounds with irreversible warhead, aiming to identifying small-molecule modulators of CRISPR-Cas9. It was found that selective inhibitors of nuclear export (SINEs) could efficiently inhibit the cellular activity of Cas9 in the form of genome-, base- and prime-editing tools. Interestingly, SINEs did not function as direct inhibitors to Cas9, but modulated Cas9 activities by interfering with the nuclear export process of Cas9 mRNA. Thus, to the best of our knowledge, SINEs represent the first reported indirect, irreversible inhibitors of CRISPR-Cas9. Most importantly, an FDA-approved anticancer drug KPT330, along with other examined SINEs, could improve the specificities of CRISPR-Cas9-based genome- and base editing tools in human cells. Our study expands the toolbox of CRISPR modulating elements and provides a feasible approach to improving the specificity of CRISPR-Cas9-based genome engineering tools.}, }
@article {pmid35301321, year = {2022}, author = {Cui, Z and Tian, R and Huang, Z and Jin, Z and Li, L and Liu, J and Huang, Z and Xie, H and Liu, D and Mo, H and Zhou, R and Lang, B and Meng, B and Weng, H and Hu, Z}, title = {FrCas9 is a CRISPR/Cas9 system with high editing efficiency and fidelity.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {1425}, pmid = {35301321}, issn = {2041-1723}, mesh = {*CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genome ; RNA, Guide/genetics ; }, abstract = {Genome editing technologies hold tremendous potential in biomedical research and drug development. Therefore, it is imperative to discover gene editing tools with superior cutting efficiency, good fidelity, and fewer genomic restrictions. Here, we report a CRISPR/Cas9 from Faecalibaculum rodentium, which is characterized by a simple PAM (5'-NNTA-3') and a guide RNA length of 21-22 bp. We find that FrCas9 could achieve comparable efficiency and specificity to SpCas9. Interestingly, the PAM of FrCas9 presents a palindromic sequence, which greatly expands its targeting scope. Due to the PAM sequence, FrCas9 possesses double editing-windows for base editor and could directly target the TATA-box in eukaryotic promoters for TATA-box related diseases. Together, our results broaden the understanding of CRISPR/Cas-mediated genome engineering and establish FrCas9 as a safe and efficient platform for wide applications in research, biotechnology and therapeutics.}, }
@article {pmid35300995, year = {2022}, author = {Hou, Y and Zhang, X and Sun, X and Qin, Q and Chen, D and Jia, M and Chen, Y}, title = {Genetically modified rabbit models for cardiovascular medicine.}, journal = {European journal of pharmacology}, volume = {922}, number = {}, pages = {174890}, doi = {10.1016/j.ejphar.2022.174890}, pmid = {35300995}, issn = {1879-0712}, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Cardiovascular Agents ; Gene Editing/methods ; Gene Targeting ; Genetic Engineering/methods ; Models, Animal ; Phenotype ; Rabbits ; }, abstract = {Genetically modified (GM) rabbits are outstanding animal models for studying human genetic and acquired diseases. As such, GM rabbits that express human genes have been extensively used as models of cardiovascular disease. Rabbits are genetically modified via prokaryotic microinjection. Through this process, genes are randomly integrated into the rabbit genome. Moreover, gene targeting in embryonic stem (ES) cells is a powerful tool for understanding gene function. However, rabbits lack stable ES cell lines. Therefore, ES-dependent gene targeting is not possible in rabbits. Nevertheless, the RNA interference technique is rapidly becoming a useful experimental tool that enables researchers to knock down specific gene expression, which leads to the genetic modification of rabbits. Recently, with the emergence of new genetic technology, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated protein 9 (CRISPR/Cas9), major breakthroughs have been made in rabbit gene targeting. Using these novel genetic techniques, researchers have successfully modified knockout (KO) rabbit models. In this paper, we aimed to review the recent advances in GM technology in rabbits and highlight their application as models for cardiovascular medicine.}, }
@article {pmid35300480, year = {2022}, author = {Lan, XR and Liu, ZL and Niu, DK}, title = {Precipitous Increase of Bacterial CRISPR-Cas Abundance at Around 45°C.}, journal = {Frontiers in microbiology}, volume = {13}, number = {}, pages = {773114}, pmid = {35300480}, issn = {1664-302X}, abstract = {Although performing adaptive immunity, CRISPR-Cas systems are present in only 40% of bacterial genomes. We observed an abrupt increase of bacterial CRISPR-Cas abundance at around 45°C. Phylogenetic comparative analyses confirmed that the abundance correlates with growth temperature only at the temperature range around 45°C. From the literature, we noticed that the diversities of cellular predators (like protozoa, nematodes, and myxobacteria) have a steep decline at this temperature range. The grazing risk faced by bacteria reduces substantially at around 45°C and almost disappears above 60°C. We propose that viral lysis would become the dominating factor of bacterial mortality, and antivirus immunity has a higher priority at higher temperatures. In temperature ranges where the abundance of cellular predators does not change with temperature, the growth temperatures of bacteria would not significantly affect their CRISPR-Cas contents. The hypothesis predicts that bacteria should also be rich in CRISPR-Cas systems if they live in other extreme conditions inaccessible to grazing predators.}, }
@article {pmid35300341, year = {2022}, author = {Xu, Y and Chen, C and Guo, Y and Hu, S and Sun, Z}, title = {Effect of CRISPR/Cas9-Edited PD-1/PD-L1 on Tumor Immunity and Immunotherapy.}, journal = {Frontiers in immunology}, volume = {13}, number = {}, pages = {848327}, pmid = {35300341}, issn = {1664-3224}, mesh = {Animals ; *B7-H1 Antigen/genetics ; CRISPR-Cas Systems ; Immunologic Factors ; Immunotherapy ; *Neoplasms/genetics/therapy ; Programmed Cell Death 1 Receptor/genetics ; }, abstract = {Clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease9 (CRISPR/Cas9) gene editing technology implements precise programming of the human genome through RNA guidance. At present, it has been widely used in the construction of animal tumor models, the study of drug resistance regulation mechanisms, epigenetic control and innovation in cancer treatment. Tumor immunotherapy restores the normal antitumor immune response by restarting and maintaining the tumor-immune cycle. CRISPR/Cas9 technology has occupied a central position in further optimizing anti-programmed cell death 1(PD-1) tumor immunotherapy. In this review, we summarize the recent progress in exploring the regulatory mechanism of tumor immune PD-1 and programmed death ligand 1(PD-L1) based on CRISPR/Cas9 technology and its clinical application in different cancer types. In addition, CRISPR genome-wide screening identifies new drug targets and biomarkers to identify potentially sensitive populations for anti-PD-1/PD-L1 therapy and maximize antitumor effects. Finally, the strong potential and challenges of CRISPR/Cas9 for future clinical applications are discussed.}, }
@article {pmid35299835, year = {2021}, author = {Parra-Flores, J and Holý, O and Bustamante, F and Lepuschitz, S and Pietzka, A and Contreras-Fernández, A and Castillo, C and Ovalle, C and Alarcón-Lavín, MP and Cruz-Córdova, A and Xicohtencatl-Cortes, J and Mancilla-Rojano, J and Troncoso, M and Figueroa, G and Ruppitsch, W}, title = {Virulence and Antibiotic Resistance Genes in Listeria monocytogenes Strains Isolated From Ready-to-Eat Foods in Chile.}, journal = {Frontiers in microbiology}, volume = {12}, number = {}, pages = {796040}, pmid = {35299835}, issn = {1664-302X}, abstract = {Listeria monocytogenes is causing listeriosis, a rare but severe foodborne infection. Listeriosis affects pregnant women, newborns, older adults, and immunocompromised individuals. Ready-to-eat (RTE) foods are the most common sources of transmission of the pathogen This study explored the virulence factors and antibiotic resistance in L. monocytogenes strains isolated from ready-to-eat (RTE) foods through in vitro and in silico testing by whole-genome sequencing (WGS). The overall positivity of L. monocytogenes in RTE food samples was 3.1% and 14 strains were isolated. L. monocytogenes ST8, ST2763, ST1, ST3, ST5, ST7, ST9, ST14, ST193, and ST451 sequence types were identified by average nucleotide identity, ribosomal multilocus sequence typing (rMLST), and core genome MLST. Seven isolates had serotype 1/2a, five 1/2b, one 4b, and one 1/2c. Three strains exhibited in vitro resistance to ampicillin and 100% of the strains carried the fosX, lin, norB, mprF, tetA, and tetC resistance genes. In addition, the arsBC, bcrBC, and clpL genes were detected, which conferred resistance to stress and disinfectants. All strains harbored hlyA, prfA, and inlA genes almost thirty-two the showed the bsh, clpCEP, hly, hpt, iap/cwhA, inlA, inlB, ipeA, lspA, mpl, plcA, pclB, oat, pdgA, and prfA genes. One isolate exhibited a type 11 premature stop codon (PMSC) in the inlA gene and another isolate a new mutation (deletion of A in position 819). The Inc18(rep25), Inc18(rep26), and N1011A plasmids and MGEs were found in nine isolates. Ten isolates showed CAS-Type II-B systems; in addition, Anti-CRISPR AcrIIA1 and AcrIIA3 phage-associated systems were detected in three genomes. These virulence and antibiotic resistance traits in the strains isolated in the RTE foods indicate a potential public health risk for consumers.}, }
@article {pmid35298777, year = {2022}, author = {Zhang, X and Tian, Y and Xu, L and Fan, Z and Cao, Y and Ma, Y and Li, H and Ren, F}, title = {CRISPR/Cas13-assisted hepatitis B virus covalently closed circular DNA detection.}, journal = {Hepatology international}, volume = {16}, number = {2}, pages = {306-315}, pmid = {35298777}, issn = {1936-0541}, support = {81770611//the National Natural Science Foundation of China/ ; 82002243//the National Natural Science Foundation of China/ ; KZ202010025035//Key Projects of the Beijing Municipal Education Commission's Science and Technology Plan/ ; 2020-1-1151//Special key research project of capital health development scientific research/ ; Z191100006619096//the Demonstrating Application and Research of Clinical Diagnosis and Treatment Technology in Beijing/ ; Z191100006619097//the Demonstrating Application and Research of Clinical Diagnosis and Treatment Technology in Beijing/ ; 2018000021469G289//Beijing Talents foundation/ ; QML20201702//Beijing Hospitals Authority Youth Programme/ ; }, mesh = {CRISPR-Cas Systems ; DNA, Circular/genetics ; DNA, Viral/analysis/genetics ; *Hepatitis B/diagnosis ; Hepatitis B virus/genetics ; *Hepatitis B, Chronic ; Humans ; Leukocytes, Mononuclear ; Real-Time Polymerase Chain Reaction/methods ; }, abstract = {BACKGROUND AND AIMS: The formation of an intranuclear pool of covalently closed circular DNA (cccDNA) in the liver is the main cause of persistent hepatitis B virus (HBV) infection. Here, we established highly sensitive and specific methods to detect cccDNA based on CRISPR-Cas13a technology.<br><br>METHODS: We used plasmid-safe ATP-dependent DNase (PSAD) enzymes and HindIII to digest loose circle rcDNA and double-stranded linear DNA, amplify specific HBV cccDNA fragments by rolling circle amplification (RCA) and PCR, and detect the target gene using CRISPR-Cas13a technology. The CRISPR-Cas13a-based assay for the detection of cccDNA was further clinically validated using HBV-related liver tissues, plasma, whole blood and peripheral blood mononuclear cells (PBMCs).<br><br>RESULTS: Based on the sample pretreatment step, the amplification step and the detection step, we established a new CRISPR-Cas13a-based assay for the detection of cccDNA. After the amplification of RCA and PCR, 1 copy/μl HBV cccDNA could be detected by CRISPR/Cas13-assisted fluorescence readout. We used ddPCR, qPCR, RCA-qPCR, PCR-CRISPR and RCA-PCR-CRISPR methods to detect 20, 4, 18, 14 and 29 positive samples in liver tissue samples from 40 HBV-related patients, respectively. HBV cccDNA was almost completely undetected in the 20 blood samples of HBV patients (including plasma, whole blood and PBMCs) by the above 5 methods.<br><br>CONCLUSIONS: We developed a novel CRISPR-based assay for the highly sensitive and specific detection of HBV cccDNA, presenting a promising alternative for accurate detection of HBV infection, antiviral therapy evaluation and treatment guidance.}, }
@article {pmid35296857, year = {2022}, author = {Li, XV and Leonardi, I and Putzel, GG and Semon, A and Fiers, WD and Kusakabe, T and Lin, WY and Gao, IH and Doron, I and Gutierrez-Guerrero, A and DeCelie, MB and Carriche, GM and Mesko, M and Yang, C and Naglik, JR and Hube, B and Scherl, EJ and Iliev, ID}, title = {Immune regulation by fungal strain diversity in inflammatory bowel disease.}, journal = {Nature}, volume = {603}, number = {7902}, pages = {672-678}, pmid = {35296857}, issn = {1476-4687}, support = {R21 AI146957/AI/NIAID NIH HHS/United States ; F32 DK120228/DK/NIDDK NIH HHS/United States ; 214229_Z_18_Z/WT_/Wellcome Trust/United Kingdom ; R37 DE022550/DE/NIDCR NIH HHS/United States ; R01 DK113136/DK/NIDDK NIH HHS/United States ; R01 DK121977/DK/NIDDK NIH HHS/United States ; R01 AI163007/AI/NIAID NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems ; Candida albicans ; *Fungi/genetics/pathogenicity ; *Gastrointestinal Microbiome ; Genetic Variation ; Humans ; Immunity ; Inflammation ; *Inflammatory Bowel Diseases ; Mammals ; *Microbiota ; *Mycobiome ; }, abstract = {The fungal microbiota (mycobiota) is an integral part of the complex multikingdom microbial community colonizing the mammalian gastrointestinal tract and has an important role in immune regulation1-6. Although aberrant changes in the mycobiota have been linked to several diseases, including inflammatory bowel disease3-9, it is currently unknown whether fungal species captured by deep sequencing represent living organisms and whether specific fungi have functional consequences for disease development in affected individuals. Here we developed a translational platform for the functional analysis of the mycobiome at the fungal-strain- and patient-specific level. Combining high-resolution mycobiota sequencing, fungal culturomics and genomics, a CRISPR-Cas9-based fungal strain editing system, in vitro functional immunoreactivity assays and in vivo models, this platform enables the examination of host-fungal crosstalk in the human gut. We discovered a rich genetic diversity of opportunistic Candida albicans strains that dominate the colonic mucosa of patients with inflammatory bowel disease. Among these human-gut-derived isolates, strains with high immune-cell-damaging capacity (HD strains) reflect the disease features of individual patients with ulcerative colitis and aggravated intestinal inflammation in vivo through IL-1β-dependent mechanisms. Niche-specific inflammatory immunity and interleukin-17A-producing T helper cell (TH17 cell) antifungal responses by HD strains in the gut were dependent on the C. albicans-secreted peptide toxin candidalysin during the transition from a benign commensal to a pathobiont state. These findings reveal the strain-specific nature of host-fungal interactions in the human gut and highlight new diagnostic and therapeutic targets for diseases of inflammatory origin.}, }
@article {pmid35294876, year = {2022}, author = {Garipler, G and Lu, C and Morrissey, A and Lopez-Zepeda, LS and Pei, Y and Vidal, SE and Zen Petisco Fiore, AP and Aydin, B and Stadtfeld, M and Ohler, U and Mahony, S and Sanjana, NE and Mazzoni, EO}, title = {The BTB transcription factors ZBTB11 and ZFP131 maintain pluripotency by repressing pro-differentiation genes.}, journal = {Cell reports}, volume = {38}, number = {11}, pages = {110524}, pmid = {35294876}, issn = {2211-1247}, support = {R01 NS100897/NS/NINDS NIH HHS/United States ; DP2 HG010099/HG/NHGRI NIH HHS/United States ; R00 HG008171/HG/NHGRI NIH HHS/United States ; R01 HD079682/HD/NICHD NIH HHS/United States ; T32 GM102057/GM/NIGMS NIH HHS/United States ; R01 GM125722/GM/NIGMS NIH HHS/United States ; R01 CA218668/CA/NCI NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; *Embryonic Stem Cells/metabolism ; Germ Layers/metabolism ; Humans ; Mice ; *Pluripotent Stem Cells/metabolism ; Transcription Factors/genetics/metabolism ; }, abstract = {In pluripotent cells, a delicate activation-repression balance maintains pro-differentiation genes ready for rapid activation. The identity of transcription factors (TFs) that specifically repress pro-differentiation genes remains obscure. By targeting ∼1,700 TFs with CRISPR loss-of-function screen, we found that ZBTB11 and ZFP131 are required for embryonic stem cell (ESC) pluripotency. ESCs without ZBTB11 or ZFP131 lose colony morphology, reduce proliferation rate, and upregulate transcription of genes associated with three germ layers. ZBTB11 and ZFP131 bind proximally to pro-differentiation genes. ZBTB11 or ZFP131 loss leads to an increase in H3K4me3, negative elongation factor (NELF) complex release, and concomitant transcription at associated genes. Together, our results suggest that ZBTB11 and ZFP131 maintain pluripotency by preventing premature expression of pro-differentiation genes and present a generalizable framework to maintain cellular potency.}, }
@article {pmid35294852, year = {2022}, author = {Gao, R and Fu, ZC and Li, X and Wang, Y and Wei, J and Li, G and Wang, L and Wu, J and Huang, X and Yang, L and Chen, J}, title = {Genomic and Transcriptomic Analyses of Prime Editing Guide RNA-Independent Off-Target Effects by Prime Editors.}, journal = {The CRISPR journal}, volume = {5}, number = {2}, pages = {276-293}, doi = {10.1089/crispr.2021.0080}, pmid = {35294852}, issn = {2573-1602}, mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; Mammals/genetics ; RNA/genetics ; *RNA, Guide/genetics ; Transcriptome ; }, abstract = {Prime editors (PEs) were developed to induce versatile edits at a guide-specified genomic locus. With all RNA-guided genome editors, guide-dependent off-target (OT) mutations can occur at other sites bearing similarity to the intended target. However, whether PEs carry the additional risk of guide-independent mutations elicited by their unique enzymatic moiety (i.e., reverse transcriptase) has not been examined systematically in mammalian cells. Here, we developed a cost-effective sensitive platform to profile guide-independent OT effects in human cells. We did not observe guide-independent OT mutations in the DNA or RNA of prime editor 3 (PE3)-edited cells, or alterations to their telomeres, endogenous retroelements, alternative splicing events, or gene expression. Together, our results showed undetectable prime editing guide RNA-independent OT effects of PE3 in human cells, suggesting the high editing specificity of its reverse-transcriptase moiety.}, }
@article {pmid35294186, year = {2022}, author = {Shi, Y and Zhang, L and Zhang, M and Chu, J and Xia, Y and Yang, H and Liu, L and Chen, X}, title = {A CRISPR-Cas9 System-Mediated Genetic Disruption and Multi-fragment Assembly in Starmerella bombicola.}, journal = {ACS synthetic biology}, volume = {11}, number = {4}, pages = {1497-1509}, doi = {10.1021/acssynbio.1c00582}, pmid = {35294186}, issn = {2161-5063}, mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Saccharomycetales/genetics ; Yeasts/genetics ; }, abstract = {Gene editing technology plays an extremely significant role in synthetic biology and metabolic engineering. Traditional genetic manipulation methods, such as homologous recombination, however, are inefficient, time-consuming, and barely feasible when disrupting multiple genes simultaneously. Starmerella bombicola, a nonconventional yeast that overproduces sophorolipids, lacks convenient genetic tools for engineering strains. Here, we developed an efficient CRISPR-Cas9 genome editing technology by combining molecular element mining and expression system optimization for S. bombicola. This CRISPR-Cas9 system improved the efficiency of gene-integration/target gene-introducing disruption by homology-directed repair and realized the multi-gene simultaneous disruptions. Based on this CRISPR-Cas9 system, we also further constructed an engineered strain via the in vivo assembly of multiple DNA fragments (10 kb) that can produce acid-type sophorolipids. These results showed that the CRISPR-Cas9 system may be an efficient and convenient strategy to perform genetic manipulation in S. bombicola.}, }
@article {pmid35292641, year = {2022}, author = {Eslami-Mossallam, B and Klein, M and Smagt, CVD and Sanden, KVD and Jones, SK and Hawkins, JA and Finkelstein, IJ and Depken, M}, title = {A kinetic model predicts SpCas9 activity, improves off-target classification, and reveals the physical basis of targeting fidelity.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {1367}, pmid = {35292641}, issn = {2041-1723}, support = {F32 AG053051/AG/NIA NIH HHS/United States ; R01 GM124141/GM/NIGMS NIH HHS/United States ; }, mesh = {*CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Endonucleases/metabolism ; Gene Editing ; RNA, Guide/genetics ; }, abstract = {The S. pyogenes (Sp) Cas9 endonuclease is an important gene-editing tool. SpCas9 is directed to target sites based on complementarity to a complexed single-guide RNA (sgRNA). However, SpCas9-sgRNA also binds and cleaves genomic off-targets with only partial complementarity. To date, we lack the ability to predict cleavage and binding activity quantitatively, and rely on binary classification schemes to identify strong off-targets. We report a quantitative kinetic model that captures the SpCas9-mediated strand-replacement reaction in free-energy terms. The model predicts binding and cleavage activity as a function of time, target, and experimental conditions. Trained and validated on high-throughput bulk-biochemical data, our model predicts the intermediate R-loop state recently observed in single-molecule experiments, as well as the associated conversion rates. Finally, we show that our quantitative activity predictor can be reduced to a binary off-target classifier that outperforms the established state-of-the-art. Our approach is extensible, and can characterize any CRISPR-Cas nuclease - benchmarking natural and future high-fidelity variants against SpCas9; elucidating determinants of CRISPR fidelity; and revealing pathways to increased specificity and efficiency in engineered systems.}, }
@article {pmid35290826, year = {2022}, author = {Nguyen, LT and Macaluso, NC and Pizzano, BLM and Cash, MN and Spacek, J and Karasek, J and Miller, MR and Lednicky, JA and Dinglasan, RR and Salemi, M and Jain, PK}, title = {A thermostable Cas12b from Brevibacillus leverages one-pot discrimination of SARS-CoV-2 variants of concern.}, journal = {EBioMedicine}, volume = {77}, number = {}, pages = {103926}, doi = {10.1016/j.ebiom.2022.103926}, pmid = {35290826}, issn = {2352-3964}, support = {U01 GH002338/GH/CGH CDC HHS/United States ; }, mesh = {*Brevibacillus/genetics ; *COVID-19/diagnosis ; Humans ; RNA, Guide ; SARS-CoV-2/genetics ; }, abstract = {BACKGROUND: Current SARS-CoV-2 detection platforms lack the ability to differentiate among variants of concern (VOCs) in an efficient manner. CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) based detection systems have the potential to transform the landscape of COVID-19 diagnostics due to their programmability; however, most of these methods are reliant on either a multi-step process involving amplification or elaborate guide RNA designs.<br><br>METHODS: Three Cas12b proteins from Alicyclobacillus acidoterrestris (AacCas12b), Alicyclobacillus acidiphilus (AapCas12b), and Brevibacillus sp. SYP-B805 (BrCas12b) were expressed and purified, and their thermostability was characterised by differential scanning fluorimetry, cis-, and trans-cleavage activities over a range of temperatures. The BrCas12b was then incorporated into a reverse transcription loop-mediated isothermal amplification (RT-LAMP)-based one-pot reaction system, coined CRISPR-SPADE (CRISPR Single Pot Assay for Detecting Emerging VOCs).<br><br>FINDINGS: Here we describe a complete one-pot detection reaction using a thermostable Cas12b effector endonuclease from Brevibacillus sp. to overcome these challenges detecting and discriminating SARS-CoV-2 VOCs in clinical samples. CRISPR-SPADE was then applied for discriminating SARS-CoV-2 VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) and validated in 208 clinical samples. CRISPR-SPADE achieved 92·8% sensitivity, 99·4% specificity, and 96·7% accuracy within 10-30 min for discriminating the SARS-CoV-2 VOCs, in agreement with S gene sequencing, achieving a positive and negative predictive value of 99·1% and 95·1%, respectively. Interestingly, for samples with high viral load (Ct value ≤ 30), 100% accuracy and sensitivity were attained. To facilitate dissemination and global implementation of the assay, a lyophilised version of one-pot CRISPR-SPADE reagents was developed and combined with an in-house portable multiplexing device capable of interpreting two orthogonal fluorescence signals.<br><br>INTERPRETATION: This technology enables real-time monitoring of RT-LAMP-mediated amplification and CRISPR-based reactions at a fraction of the cost of a qPCR system. The thermostable Brevibacillus sp. Cas12b offers relaxed primer design for accurately detecting SARS-CoV-2 VOCs in a simple and robust one-pot assay. The lyophilised reagents and simple instrumentation further enable rapid deployable point-of-care diagnostics that can be easily expanded beyond COVID-19.<br><br>FUNDING: This project was funded in part by the United States-India Science &amp; Technology Endowment Fund- COVIDI/247/2020 (P.K.J.), Florida Breast Cancer Foundation- AGR00018466 (P.K.J.), National Institutes of Health- NIAID 1R21AI156321-01 (P.K.J.), Centers for Disease Control and Prevention- U01GH002338 (R.R.D., J.A.L., &amp; P.K.J.), University of Florida, Herbert Wertheim College of Engineering (P.K.J.), University of Florida Vice President Office of Research and CTSI seed funds (M.S.), and University of Florida College of Veterinary Medicine and Emerging Pathogens Institute (R.R.D.).}, }
@article {pmid35290629, year = {2022}, author = {Chen, BR and Sleckman, BP}, title = {A Whole Genome CRISPR/Cas9 Screening Approach for Identifying Genes Encoding DNA End-Processing Proteins.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2444}, number = {}, pages = {15-27}, pmid = {35290629}, issn = {1940-6029}, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; DNA ; DNA Breaks, Double-Stranded ; *DNA End-Joining Repair ; Mice ; RNA, Guide/genetics ; }, abstract = {DNA double-strand breaks (DSBs) are mainly repaired by homologous recombination (HR) and non-homologous end joining (NHEJ). The choice of HR or NHEJ is dictated in part by whether the broken DNA ends are resected to generate extended single-stranded DNA (ssDNA) overhangs, which are quickly bound by the trimeric ssDNA binding complex RPA, the first step of HR. Here we describe a series of protocols for generating Abelson murine leukemia virus-transformed pre-B cells (abl pre-B cells) with stably integrated inducible Cas9 that can be used to identify and study novel pathways regulating DNA end processing. These approaches involve gene inactivation by CRISPR/Cas9, whole genome guide RNA (gRNA) library-mediated screen, and flow cytometry-based detection of chromatin-bound RPA after DNA damage.}, }
@article {pmid35290572, year = {2022}, author = {Wei, W and Gao, C}, title = {Gene editing: from technologies to applications in research and beyond.}, journal = {Science China. Life sciences}, volume = {65}, number = {4}, pages = {657-659}, pmid = {35290572}, issn = {1869-1889}, mesh = {*CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Technology ; }, }
@article {pmid35289346, year = {2022}, author = {Zhou, J and Hu, J and Liu, R and Wang, C and Lv, Y}, title = {Dual-amplified CRISPR-Cas12a bioassay for HIV-related nucleic acids.}, journal = {Chemical communications (Cambridge, England)}, volume = {58}, number = {26}, pages = {4247-4250}, doi = {10.1039/d2cc00792d}, pmid = {35289346}, issn = {1364-548X}, mesh = {Biological Assay ; CRISPR-Cas Systems/genetics ; Gold ; *HIV Infections ; Humans ; *Metal Nanoparticles ; *Nucleic Acids ; }, abstract = {Nucleic acid amplification strategies have successfully dominated ultrasensitive bioassays, but they sometimes bring high time-consumption, multi-step operation, increased contamination risk, and mismatch-related inaccuracy. We proposed a nucleic acid amplification-free method called the AuNPs-tagging based CRISPR-Cas12a bioassay platform. The signal amplification was realized by integrating the self-amplification effect of CRISPR-Cas12a with the enhancement effect of the large number of detectable atoms inside each gold nanoparticle. The proposed method achieved a low LOD of 1.05 amol in 40 min for HIV-related DNA.}, }
@article {pmid35288985, year = {2022}, author = {Kabay, G and DeCastro, J and Altay, A and Smith, K and Lu, HW and Capossela, AM and Moarefian, M and Aran, K and Dincer, C}, title = {Emerging Biosensing Technologies for the Diagnostics of Viral Infectious Diseases.}, journal = {Advanced materials (Deerfield Beach, Fla.)}, volume = {}, number = {}, pages = {e2201085}, doi = {10.1002/adma.202201085}, pmid = {35288985}, issn = {1521-4095}, support = {404478562//Deutsche Forschungsgemeinschaft/ ; 421356369//Deutsche Forschungsgemeinschaft/ ; 446617142//Deutsche Forschungsgemeinschaft/ ; 13GW0493//Bundesministerium für Bildung und Forschung/ ; 2048283//NSF/ ; R01 HL139605/GF/NIH HHS/United States ; GR700029RDA//Keck Start-up to Aran Lab/ ; }, abstract = {Several viral infectious diseases appear limitless since the beginning of the 21st century, expanding into pandemic lengths. Thus, there are extensive efforts to provide more efficient means of diagnosis, a better understanding of acquired immunity, and improved monitoring of inflammatory biomarkers, as these are all crucial for controlling the spread of infection while aiding in vaccine development and improving patient outcomes. In this regard, various biosensors have been developed recently to streamline pathogen and immune response detection by addressing the limitations of traditional methods, including isothermal amplification-based systems and lateral flow assays. This review explores state-of-the-art biosensors for detecting viral pathogens, serological assays, and inflammatory biomarkers from the material perspective, by discussing their advantages, limitations, and further potential regarding their analytical performance, clinical utility, and point-of-care adaptability. Additionally, next-generation biosensing technologies that offer better sensitivity and selectivity, and easy handling for end-users are highlighted. An emerging example of these next-generation biosensors are those powered by novel synthetic biology tools, such as clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated proteins (Cas), in combination with integrated point-of-care devices. Lastly, the current challenges are discussed and a roadmap for furthering these advanced biosensing technologies to manage future pandemics is provided.}, }
@article {pmid35288718, year = {2022}, author = {Kaltenbacher, T and Löprich, J and Maresch, R and Weber, J and Müller, S and Oellinger, R and Groß, N and Griger, J and de Andrade Krätzig, N and Avramopoulos, P and Ramanujam, D and Brummer, S and Widholz, SA and Bärthel, S and Falcomatà, C and Pfaus, A and Alnatsha, A and Mayerle, J and Schmidt-Supprian, M and Reichert, M and Schneider, G and Ehmer, U and Braun, CJ and Saur, D and Engelhardt, S and Rad, R}, title = {CRISPR somatic genome engineering and cancer modeling in the mouse pancreas and liver.}, journal = {Nature protocols}, volume = {17}, number = {4}, pages = {1142-1188}, pmid = {35288718}, issn = {1750-2799}, support = {DFG RA 1629/2-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; SA1374/4-2//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 70114314//Deutsche Krebshilfe (German Cancer Aid)/ ; }, mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/methods ; Liver ; Mice ; Mice, Knockout ; *Neoplasms/genetics ; Pancreas ; }, abstract = {Genetically engineered mouse models (GEMMs) transformed the study of organismal disease phenotypes but are limited by their lengthy generation in embryonic stem cells. Here, we describe methods for rapid and scalable genome engineering in somatic cells of the liver and pancreas through delivery of CRISPR components into living mice. We introduce the spectrum of genetic tools, delineate viral and nonviral CRISPR delivery strategies and describe a series of applications, ranging from gene editing and cancer modeling to chromosome engineering or CRISPR multiplexing and its spatio-temporal control. Beyond experimental design and execution, the protocol describes quantification of genetic and functional editing outcomes, including sequencing approaches, data analysis and interpretation. Compared to traditional knockout mice, somatic GEMMs face an increased risk for mouse-to-mouse variability because of the higher experimental demands of the procedures. The robust protocols described here will help unleash the full potential of somatic genome manipulation. Depending on the delivery method and envisaged application, the protocol takes 3-5 weeks.}, }
@article {pmid35288688, year = {2022}, author = {Shaffer, C}, title = {Broad defeats Berkeley CRISPR patent.}, journal = {Nature biotechnology}, volume = {40}, number = {4}, pages = {445}, doi = {10.1038/d41587-022-00004-2}, pmid = {35288688}, issn = {1546-1696}, mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; }, }
@article {pmid35288582, year = {2022}, author = {Biegler, MT and Fedrigo, O and Collier, P and Mountcastle, J and Haase, B and Tilgner, HU and Jarvis, ED}, title = {Induction of an immortalized songbird cell line allows for gene characterization and knockout by CRISPR-Cas9.}, journal = {Scientific reports}, volume = {12}, number = {1}, pages = {4369}, pmid = {35288582}, issn = {2045-2322}, support = {R01 GM135247/GM/NIGMS NIH HHS/United States ; RF1 MH121267/MH/NIMH NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; U01 DA053625/DA/NIDA NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; *Finches/genetics ; Genome ; Genomics ; }, abstract = {The zebra finch is one of the most commonly studied songbirds in biology, particularly in genomics, neuroscience and vocal communication. However, this species lacks a robust cell line for molecular biology research and reagent optimization. We generated a cell line, designated CFS414, from zebra finch embryonic fibroblasts using the SV40 large and small T antigens. This cell line demonstrates an improvement over previous songbird cell lines through continuous and density-independent growth, allowing for indefinite culture and monoclonal line derivation. Cytogenetic, genomic, and transcriptomic profiling established the provenance of this cell line and identified the expression of genes relevant to ongoing songbird research. Using this cell line, we disrupted endogenous gene sequences using S.aureus Cas9 and confirmed a stress-dependent localization response of a song system specialized gene, SAP30L. The utility of CFS414 cells enhances the comprehensive molecular potential of the zebra finch and validates cell immortalization strategies in a songbird species.}, }
@article {pmid35288574, year = {2022}, author = {Sangree, AK and Griffith, AL and Szegletes, ZM and Roy, P and DeWeirdt, PC and Hegde, M and McGee, AV and Hanna, RE and Doench, JG}, title = {Benchmarking of SpCas9 variants enables deeper base editor screens of BRCA1 and BCL2.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {1318}, pmid = {35288574}, issn = {2041-1723}, mesh = {Benchmarking ; *CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Proto-Oncogene Proteins c-bcl-2/genetics ; }, abstract = {Numerous rationally-designed and directed-evolution variants of SpCas9 have been reported to expand the utility of CRISPR technology. Here, we assess the activity and specificity of WT-Cas9 and 10 SpCas9 variants by benchmarking their PAM preferences, on-target activity, and off-target susceptibility in cell culture assays with thousands of guides targeting endogenous genes. To enhance the coverage and thus utility of base editing screens, we demonstrate that the SpCas9-NG and SpG variants are compatible with both A > G and C > T base editors, more than tripling the number of guides and assayable residues. We demonstrate the performance of these technologies by screening for loss-of-function mutations in BRCA1 and Venetoclax-resistant mutations in BCL2, identifying both known and new mutations that alter function. We anticipate that the tools and methodologies described here will facilitate the investigation of genetic variants at a finer and deeper resolution for any locus of interest.}, }
@article {pmid35288457, year = {2022}, author = {Ding, L and Schmitt, LT and Brux, M and Sürün, D and Augsburg, M and Lansing, F and Mircetic, J and Theis, M and Buchholz, F}, title = {DNA methylation-independent long-term epigenetic silencing with dCRISPR/Cas9 fusion proteins.}, journal = {Life science alliance}, volume = {5}, number = {6}, pages = {}, pmid = {35288457}, issn = {2575-1077}, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *DNA Methylation/genetics ; Epigenesis, Genetic/genetics ; Gene Editing/methods ; HEK293 Cells ; Humans ; Mice ; RNA, Guide/genetics ; }, abstract = {The programmable CRISPR/Cas9 DNA nuclease is a versatile genome editing tool, but it requires the host cell DNA repair machinery to alter genomic sequences. This fact leads to unpredictable changes of the genome at the cut sites. Genome editing tools that can alter the genome without causing DNA double-strand breaks are therefore in high demand. Here, we show that expression of promoter-associated short guide (sg)RNAs together with dead Cas9 (dCas9) fused to a Krüppel-associated box domains (KRABd) in combination with the transcription repression domain of methyl CpG-binding protein 2 (MeCP2) can lead to persistent gene silencing in mouse embryonic stem cells and in human embryonic kidney (HEK) 293 cells. Surprisingly, this effect is achievable and even enhanced in DNA (cytosine-5)-methyltransferase 3A and 3B (Dnmt3A-/-, Dnmt3b-/-) depleted cells. Our results suggest that dCas9-KRABd-MeCP2 fusions are useful for long-term epigenetic gene silencing with utility in cell biology and potentially in therapeutical settings.}, }
@article {pmid35288230, year = {2022}, author = {Zheng, SY and Ma, LL and Wang, XL and Lu, LX and Ma, ST and Xu, B and Ouyang, W}, title = {RPA-Cas12aDS: A visual and fast molecular diagnostics platform based on RPA-CRISPR-Cas12a method for infectious bursal disease virus detection.}, journal = {Journal of virological methods}, volume = {304}, number = {}, pages = {114523}, doi = {10.1016/j.jviromet.2022.114523}, pmid = {35288230}, issn = {1879-0984}, mesh = {Animals ; *Birnaviridae Infections/diagnosis/veterinary ; CRISPR-Cas Systems ; Chickens ; *Infectious bursal disease virus/genetics ; Nucleic Acid Amplification Techniques/methods ; Pathology, Molecular ; *Poultry Diseases/diagnosis ; Recombinases/genetics ; }, abstract = {Infectious bursal disease (IBD), a major disease of birds, is caused by infectious bursal disease virus (IBDV). The disease can lead to immunosuppression, resulting in huge economic losses in the poultry industry. A specific, rapid, and simple detection method is important for the early diagnosis and prevention and control of IBDV. In this study, we established a naked-eye visual IBDV detection method, named "RPA-Cas12aDS", by combining recombinase polymerase amplification (RPA) with CRISPR-Cas12a-based nucleic acid detection. The detection process can be accomplished in 50 min, and uncapping contamination can be avoided. The detection results can be observed under blue or UV light. We used the RPA-Cas12aDS method to detect IBDV in bursa of Fabricius tissue samples of chickens, and the results were consistent with those obtained using commercial RT-PCR kits. This method presents great potential for visual, rapid, and point-of-care molecular diagnostics of IBDV in poultry.}, }
@article {pmid35286975, year = {2022}, author = {Hommersom, MP and Bijnagte-Schoenmaker, C and Albert, S and van de Warrenburg, BPC and Nadif Kasri, N and van Bokhoven, H}, title = {Generation of induced pluripotent stem cell lines carrying monoallelic (UCSFi001-A-60) or biallelic (UCSFi001-A-61; UCSFi001-A-62) frameshift variants in CACNA1A using CRISPR/Cas9.}, journal = {Stem cell research}, volume = {61}, number = {}, pages = {102730}, doi = {10.1016/j.scr.2022.102730}, pmid = {35286975}, issn = {1876-7753}, mesh = {CRISPR-Cas Systems/genetics ; Calcium Channels/metabolism ; Cells, Cultured ; Frameshift Mutation ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; }, abstract = {CACNA1A encodes a P/Q-type voltage-gated calcium channel. Heterozygous loss-of-function variants in this gene have been associated with episodic ataxia type 2. In this study, we used CRISPR/Cas9 to generate isogenic human induced pluripotent stem cell lines with a gene-dosage dependent deficiency of CACNA1A. We obtained one clone with monoallelic (UCSFi001-A-60) and two clones with biallelic (UCSFi001-A-61; UCSFi001-A-62) frameshift variants in CACNA1A. All three lines showed expression of pluripotency markers and a normal karyotype.}, }
@article {pmid35286377, year = {2022}, author = {Pallaseni, A and Peets, EM and Koeppel, J and Weller, J and Vanderstichele, T and Ho, UL and Crepaldi, L and van Leeuwen, J and Allen, F and Parts, L}, title = {Predicting base editing outcomes using position-specific sequence determinants.}, journal = {Nucleic acids research}, volume = {50}, number = {6}, pages = {3551-3564}, pmid = {35286377}, issn = {1362-4962}, support = {/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; 206194/WT_/Wellcome Trust/United Kingdom ; }, mesh = {Adenine ; *CRISPR-Cas Systems ; Cytosine/metabolism ; *Gene Editing ; Humans ; Nucleotides ; }, abstract = {CRISPR/Cas base editors promise nucleotide-level control over DNA sequences, but the determinants of their activity remain incompletely understood. We measured base editing frequencies in two human cell lines for two cytosine and two adenine base editors at ∼14 000 target sequences and find that base editing activity is sequence-biased, with largest effects from nucleotides flanking the target base. Whether a base is edited depends strongly on the combination of its position in the target and the preceding base, acting to widen or narrow the effective editing window. The impact of features on editing rate depends on the position, with sequence bias efficacy mainly influencing bases away from the center of the window. We use these observations to train a machine learning model to predict editing activity per position, with accuracy ranging from 0.49 to 0.72 between editors, and with better generalization across datasets than existing tools. We demonstrate the usefulness of our model by predicting the efficacy of disease mutation correcting guides, and find that most of them suffer from more unwanted editing than pure outcomes. This work unravels the position-specificity of base editing biases and allows more efficient planning of editing campaigns in experimental and therapeutic contexts.}, }
@article {pmid35286371, year = {2022}, author = {Xiong, X and Li, Z and Liang, J and Liu, K and Li, C and Li, JF}, title = {A cytosine base editor toolkit with varying activity windows and target scopes for versatile gene manipulation in plants.}, journal = {Nucleic acids research}, volume = {50}, number = {6}, pages = {3565-3580}, pmid = {35286371}, issn = {1362-4962}, mesh = {Adenine ; Animals ; *CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Cytosine ; *Gene Editing ; }, abstract = {CRISPR/Cas-derived base editing tools empower efficient alteration of genomic cytosines or adenines associated with essential genetic traits in plants and animals. Diversified target sequences and customized editing products call for base editors with distinct features regarding the editing window and target scope. Here we developed a toolkit of plant base editors containing AID10, an engineered human AID cytosine deaminase. When fused to the N-terminus or C-terminus of the conventional Cas9 nickase (nSpCas9), AID10 exhibited a broad or narrow activity window at the protospacer adjacent motif (PAM)-distal and -proximal protospacer, respectively, while AID10 fused to both termini conferred an additive activity window. We further replaced nSpCas9 with orthogonal or PAM-relaxed Cas9 variants to widen target scopes. Moreover, we devised dual base editors with AID10 located adjacently or distally to the adenine deaminase ABE8e, leading to juxtaposed or spaced cytosine and adenine co-editing at the same target sequence in plant cells. Furthermore, we expanded the application of this toolkit in plants for tunable knockdown of protein-coding genes via creating upstream open reading frame and for loss-of-function analysis of non-coding genes, such as microRNA sponges. Collectively, this toolkit increases the functional diversity and versatility of base editors in basic and applied plant research.}, }
@article {pmid35285719, year = {2022}, author = {Xu, X and Harvey-Samuel, T and Siddiqui, HA and Ang, JX and Anderson, ME and Reitmayer, CM and Lovett, E and Leftwich, PT and You, M and Alphey, L}, title = {Toward a CRISPR-Cas9-Based Gene Drive in the Diamondback Moth Plutella xylostella.}, journal = {The CRISPR journal}, volume = {5}, number = {2}, pages = {224-236}, doi = {10.1089/crispr.2021.0129}, pmid = {35285719}, issn = {2573-1602}, support = {BB/S506680/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007033/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007038/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007039/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; }, mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Gene Drive Technology ; Gene Editing ; Larva/genetics/metabolism ; Mice ; *Moths/genetics ; }, abstract = {Promising to provide powerful genetic control tools, gene drives have been constructed in multiple dipteran insects, yeast, and mice for the purposes of population elimination or modification. However, it remains unclear whether these techniques can be applied to lepidopterans. Here, we used endogenous regulatory elements to drive Cas9 and single guide RNA (sgRNA) expression in the diamondback moth (DBM), Plutella xylostella, and test the first split gene drive system in a lepidopteran. The DBM is an economically important global agriculture pest of cruciferous crops and has developed severe resistance to various insecticides, making it a prime candidate for such novel control strategy development. A very high level of somatic editing was observed in Cas9/sgRNA transheterozygotes, although no significant homing was revealed in the subsequent generation. Although heritable Cas9-medated germline cleavage as well as maternal and paternal Cas9 deposition were observed, rates were far lower than for somatic cleavage events, indicating robust somatic but limited germline activity of Cas9/sgRNA under the control of selected regulatory elements. Our results provide valuable experience, paving the way for future construction of gene drives or other Cas9-based genetic control strategies in DBM and other lepidopterans.}, }
@article {pmid35285707, year = {2022}, author = {Parcey, M and Gayder, S and Castle, AJ and Svircev, AM}, title = {Function and Application of the CRISPR-Cas System in the Plant Pathogen Erwinia amylovora.}, journal = {Applied and environmental microbiology}, volume = {88}, number = {7}, pages = {e0251321}, pmid = {35285707}, issn = {1098-5336}, mesh = {*Bacteriophages/genetics ; CRISPR-Cas Systems ; *Erwinia amylovora/genetics ; Plasmids/genetics ; Streptomycin ; }, abstract = {Phage-based biocontrol is an emerging method for managing the plant pathogen Erwinia amylovora. Control of E. amylovora in North America is achieved chiefly through the application of streptomycin and has led to the development of streptomycin resistance. Resistant E. amylovora can be tracked through the analysis of CRISPR spacer sequences. An alternative to antibiotics are bacterial viruses, known as phages, which lyse their hosts during replication to control the bacterial population. Endogenous CRISPR-Cas systems act as phage resistance mechanisms however, preliminary genomic analysis suggests this activity is limited in E. amylovora. This leaves the functionality of the CRISPR-Cas system, any clade-based differences, and the impact which this system may have on phage-based biocontrol in question. In this study, the CRISPR arrays from 127 newly available genomic sequences of E. amylovora were analyzed through a novel bioinformatic pipeline. Through this, the Eastern and Western North American clades were shown to be incompatible with the current PCR-based approaches for tracking E. amylovora given the size and composition of their CRISPR arrays. Two artificial CRISPR arrays were designed to investigate the functionality of the CRISPR-Cas system in E. amylovora. This system was capable of curing a targeted plasmid and providing phage resistance but was not the source of phage resistance observed within the controls. This suggests that while the CRISPR-Cas system is an important defense mechanism for invasive plasmids, an as yet unidentified mechanism is the primary source of phage resistance in E. amylovora. IMPORTANCE Erwinia amylovora is an economically significant agricultural pathogen found throughout the world. In North America, E. amylovora has developed streptomycin resistance and therefore alternative treatments using phages have received increased attention. In this study, we analyzed recently published genomes to determine that two significant groups of E. amylovora are poorly identified using the current, CRISPR-based tracking methods. We also showed that the CRISPR-Cas system and an unidentified mechanism work together to provide a significant degree of resistance against one of the phages proposed for phage-based biocontrol.}, }
@article {pmid35284843, year = {2022}, author = {Aouida, M and Aljogol, D and Ali, R and Ramotar, D}, title = {A simple protocol to isolate a single human cell PRDX1 knockout generated by CRISPR-Cas9 system.}, journal = {STAR protocols}, volume = {3}, number = {1}, pages = {101216}, pmid = {35284843}, issn = {2666-1667}, mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Gene Knockout Techniques ; Humans ; Peroxiredoxins/genetics ; *RNA, Guide/genetics ; Transfection ; }, abstract = {Here, we describe a protocol for human PRDX1 gene knockout cells using the CRISPR-Cas9 system. The protocol describes all the steps sequentially: (1) single-guide RNA design, cloning, and transfection; (2) gene editing evaluation by T7EI assay; (3) single-cell isolation; and (4) knockout verification to determine indels in one or both alleles by Sanger sequencing. This strategy is based on the efficiency of DNA editing, avoids antibiotic selection, and bypasses the need for cell sorting.}, }
@article {pmid35284835, year = {2022}, author = {Oh, S and Buisson, R}, title = {A digital PCR-based protocol to detect and quantify RNA editing events at hotspots.}, journal = {STAR protocols}, volume = {3}, number = {1}, pages = {101148}, pmid = {35284835}, issn = {2666-1667}, support = {P30 CA062203/CA/NCI NIH HHS/United States ; R00 CA212154/CA/NCI NIH HHS/United States ; R37 CA252081/CA/NCI NIH HHS/United States ; }, mesh = {CRISPR-Cas Systems ; Cytidine Deaminase ; *Gene Editing/methods ; Humans ; Polymerase Chain Reaction ; Proteins ; RNA ; *RNA Editing/genetics ; }, abstract = {APOBEC3A, CRISPR programmable RNA base editors, or other enzymes can edit RNA transcripts at specific locations or hotspots. Precise quantification of these RNA-editing events is crucial to determine the activity and efficiency of these enzymes in cells. We have developed a quick method to quantify RNA-editing activity using digital PCR, a sensitive and quantitative technique to detect rare mutations by micro-partitioning bulk PCR reactions. This assay allows rapid absolute quantification of RNA editing events in cell lines or patient samples. For complete details on the use and execution of this protocol, please refer to Jalili et al. (2020) and Oh et al. (2021).}, }
@article {pmid35284406, year = {2022}, author = {Chen, B and Li, Y and Xu, F and Yang, X}, title = {Powerful CRISPR-Based Biosensing Techniques and Their Integration With Microfluidic Platforms.}, journal = {Frontiers in bioengineering and biotechnology}, volume = {10}, number = {}, pages = {851712}, pmid = {35284406}, issn = {2296-4185}, abstract = {In the fight against the worldwide pandemic coronavirus disease 2019 (COVID-19), simple, rapid, and sensitive tools for nucleic acid detection are in urgent need. PCR has been a classic method for nucleic acid detection with high sensitivity and specificity. However, this method still has essential limitations due to the dependence on thermal cycling, which requires costly equipment, professional technicians, and long turnover times. Currently, clustered regularly interspaced short palindromic repeats (CRISPR)-based biosensors have been developed as powerful tools for nucleic acid detection. Moreover, the CRISPR method can be performed at physiological temperature, meaning that it is easy to assemble into point-of-care devices. Microfluidic chips hold promises to integrate sample processing and analysis on a chip, reducing the consumption of sample and reagent and increasing the detection throughput. This review provides an overview of recent advances in the development of CRISPR-based biosensing techniques and their perfect combination with microfluidic platforms. New opportunities and challenges for the improvement of specificity and efficiency signal amplification are outlined. Furthermore, their various applications in healthcare, animal husbandry, agriculture, and forestry are discussed.}, }
@article {pmid35283690, year = {2022}, author = {Ivanova, E}, title = {How Various Drug Delivery Methods Could Aid in the Translation of Genome Prime Editing Technologies.}, journal = {Genetics research}, volume = {2022}, number = {}, pages = {7301825}, pmid = {35283690}, issn = {1469-5073}, mesh = {*CRISPR-Cas Systems ; Drug Delivery Systems ; *Gene Editing/methods ; Pharmaceutical Preparations ; Technology ; }, abstract = {Drug delivery systems can be engineered to enhance the localization of therapeutics in specific tissues in response to externally applied stimuli and/or local environmental changes. In recent decades, efforts to improve drug delivery techniques at both nano- and macroscale have led to a new era of therapeutic efficacy. Such technological advancements resulted in improved drug delivery systems regularly entering the clinical setting. However, these delivery innovations are unfortunately not always readily applied to newly developed technologies. One of these new and exciting technologies that has been overlooked by drug delivery scientists is prime editing. Prime editing is a novel genome editing technology that exhibits the plug-and-play capability of CRISPR/Cas9 editors while avoiding double-strand DNA breaks throughout the entire process. This article focuses on describing the potential advantages and disadvantages of selecting nanomedicine technologies along with prime editing capabilities for the delivery of cargo.}, }
@article {pmid35281441, year = {2022}, author = {Garre, V}, title = {Recent Advances and Future Directions in the Understanding of Mucormycosis.}, journal = {Frontiers in cellular and infection microbiology}, volume = {12}, number = {}, pages = {850581}, pmid = {35281441}, issn = {2235-2988}, mesh = {Antifungal Agents/therapeutic use ; *COVID-19 ; Genomics ; Humans ; *Mucorales/genetics ; *Mucormycosis/diagnosis/microbiology/pathology ; }, abstract = {Mucormycosis is an emerging infection caused by fungi of the order Mucorales that has recently gained public relevance due to the high incidence among COVID-19 patients in some countries. The reduced knowledge about Mucorales pathogenesis is due, in large part, to the historically low interest for these fungi fostered by their reluctance to be genetically manipulated. The recent introduction of more tractable genetic models together with an increasing number of available whole genome sequences and genomic analyses have improved our understanding of Mucorales biology and mucormycosis in the last ten years. This review summarizes the most significant advances in diagnosis, understanding of the innate and acquired resistance to antifungals, identification of new virulence factors and molecular mechanisms involved in the infection. The increased awareness about the disease and the recent successful genetic manipulation of previous intractable fungal models using CRISPR-Cas9 technology are expected to fuel the characterization of Mucorales pathogenesis, facilitating the development of effective treatments to fight this deadly infection.}, }
@article {pmid35279545, year = {2022}, author = {de Toledo, MAS and Fu, X and Kluge, F and Götz, K and Schmitz, S and Wanek, P and Schüler, HM and Pannen, K and Chatain, N and Koschmieder, S and Brümmendorf, TH and Zenke, M}, title = {CRISPR/Cas9-engineered human ES cells harboring heterozygous and homozygous c-KIT knockout.}, journal = {Stem cell research}, volume = {60}, number = {}, pages = {102732}, doi = {10.1016/j.scr.2022.102732}, pmid = {35279545}, issn = {1876-7753}, mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Heterozygote ; Homozygote ; *Human Embryonic Stem Cells/metabolism ; Humans ; }, abstract = {The receptor tyrosine kinase c-KIT (CD117) has a key role in hematopoiesis and is a marker for endothelial and cardiac progenitor cells. In vivo, deficiency of c-KIT is lethal and therefore using CRISPR/Cas9 editing we generated heterozygous and homozygous c-KIT knockout human embryonic stem cell (ES cell) lines. The c-KIT knockout left ES cell pluripotency unaffected as shown by immunofluorescence and trilineage differentiation potential. Heterozygous and homozygous c-KIT knockouts showed complete loss of exon 17, resulting in ablation of c-KIT protein from the cell surface. c-KIT knockout ES cells provide a valuable tool for further investigating c-KIT biology.}, }
@article {pmid35278851, year = {2022}, author = {Xie, Z and Chen, S and Zhang, W and Zhao, S and Zhao, Z and Wang, X and Huang, Y and Yi, G}, title = {A novel fluorescence amplification strategy combining cascade primer exchange reaction with CRISPR/Cas12a system for ultrasensitive detection of RNase H activity.}, journal = {Biosensors &amp; bioelectronics}, volume = {206}, number = {}, pages = {114135}, doi = {10.1016/j.bios.2022.114135}, pmid = {35278851}, issn = {1873-4235}, mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; DNA/genetics/metabolism ; Humans ; Nucleic Acid Amplification Techniques/methods ; RNA ; Ribonuclease H ; }, abstract = {Ribonuclease H (RNase H), which plays a vital role in various cellular processes, is to be closely related to the emergence of many diseases. As an essential therapeutic target, it shows great prospects in the development of associated drugs. Herein, a DNA-RNA chimeric hairpin (DR HP) was designed to introduce a new signal amplification strategy based on cascade primer exchange reaction (cPER) and CRISPR/Cas12a system for sensitive and specific analysis of RNase H activity. In the presence of RNase H, the RNA fragment of DR HP was specifically degraded and the blocked primer DNA was released. The process of enzymatic hydrolysis of substrate hairpin and cyclic signal amplification was completed in a one-step method under isothermal conditions, enriching many activator strands to initiate trans-cleavage of CRISPR/Cas system, thereby restoring the fluorescence signal. Under optimized conditions, the developed strategy exhibited a good linear relationship ranging from 0.005 to 0.1U/mL and offered a detection limit of 0.00061U/mL. Moreover, this method was used for RNase H activity assay in complicated human serum and real cell lysates with good stability and repeatability, and was also demonstrated to apply for RNase H inhibitors screening and inhibitory capability assessment. Therefore, the proposed system is a promising platform not only for determination of RNase H activity, but open up new thoughts for the biological enzyme research and inhibitor screening.}, }
@article {pmid35277676, year = {2022}, author = {Mullard, A}, title = {Proof-of-principle Intellia trial shows in vivo CRISPR activity.}, journal = {Nature reviews. Drug discovery}, volume = {21}, number = {4}, pages = {249}, doi = {10.1038/d41573-022-00050-0}, pmid = {35277676}, issn = {1474-1784}, mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; Humans ; }, }
@article {pmid35277177, year = {2022}, author = {Lan, T and Que, H and Luo, M and Zhao, X and Wei, X}, title = {Genome editing via non-viral delivery platforms: current progress in personalized cancer therapy.}, journal = {Molecular cancer}, volume = {21}, number = {1}, pages = {71}, pmid = {35277177}, issn = {1476-4598}, mesh = {CRISPR-Cas Systems ; *Gene Editing ; Genes, Neoplasm ; Genetic Therapy ; Humans ; *Neoplasms/genetics/therapy ; }, abstract = {Cancer is a severe disease that substantially jeopardizes global health. Although considerable efforts have been made to discover effective anti-cancer therapeutics, the cancer incidence and mortality are still growing. The personalized anti-cancer therapies present themselves as a promising solution for the dilemma because they could precisely destroy or fix the cancer targets based on the comprehensive genomic analyses. In addition, genome editing is an ideal way to implement personalized anti-cancer therapy because it allows the direct modification of pro-tumor genes as well as the generation of personalized anti-tumor immune cells. Furthermore, non-viral delivery system could effectively transport genome editing tools (GETs) into the cell nucleus with an appreciable safety profile. In this manuscript, the important attributes and recent progress of GETs will be discussed. Besides, the laboratory and clinical investigations that seek for the possibility of combining non-viral delivery systems with GETs for the treatment of cancer will be assessed in the scope of personalized therapy.}, }
@article {pmid35276091, year = {2022}, author = {Simkin, D and Papakis, V and Bustos, BI and Ambrosi, CM and Ryan, SJ and Baru, V and Williams, LA and Dempsey, GT and McManus, OB and Landers, JE and Lubbe, SJ and George, AL and Kiskinis, E}, title = {Homozygous might be hemizygous: CRISPR/Cas9 editing in iPSCs results in detrimental on-target defects that escape standard quality controls.}, journal = {Stem cell reports}, volume = {17}, number = {4}, pages = {993-1008}, pmid = {35276091}, issn = {2213-6711}, mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Homozygote ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Quality Control ; }, abstract = {The ability to precisely edit the genome of human induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9 has enabled the development of cellular models that can address genotype to phenotype relationships. While genome editing is becoming an essential tool in iPSC-based disease modeling studies, there is no established quality control workflow for edited cells. Moreover, large on-target deletions and insertions that occur through DNA repair mechanisms have recently been uncovered in CRISPR/Cas9-edited loci. Yet the frequency of these events in human iPSCs remains unclear, as they can be difficult to detect. We examined 27 iPSC clones generated after targeting 9 loci and found that 33% had acquired large, on-target genomic defects, including insertions and loss of heterozygosity. Critically, all defects had escaped standard PCR and Sanger sequencing analysis. We describe a cost-efficient quality control strategy that successfully identified all edited clones with detrimental on-target events and could facilitate the integrity of iPSC-based studies.}, }
@article {pmid35271371, year = {2022}, author = {Dominguez, AA and Chavez, MG and Urke, A and Gao, Y and Wang, L and Qi, LS}, title = {CRISPR-Mediated Synergistic Epigenetic and Transcriptional Control.}, journal = {The CRISPR journal}, volume = {5}, number = {2}, pages = {264-275}, doi = {10.1089/crispr.2021.0099}, pmid = {35271371}, issn = {2573-1602}, support = {U01 DK127405/DK/NIDDK NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Epigenomics ; Gene Editing ; Gene Expression Regulation/genetics ; }, abstract = {Targeted activation of endogenous genes is an important approach for cell engineering. Here, we report that the nuclease-deactivated dCas9 fused to a transcriptional activator (VPR) and an epigenetic effector (the catalytic domain of histone acetyltransferase p300core) simultaneously, sequentially, or as a single quadripartite effector can lead to enhanced activation of target genes. The composite activator, VPRP, behaves more efficiently than individual activators across a set of genes in different cell types. We characterize off-target effects for host chromatin acetylation and transcriptome using the effectors. Our work demonstrates that transcriptional and epigenetic effectors can be used together to enhance gene activation and suggests the need for further optimization of epigenetic effectors to reduce off-targets.}, }
@article {pmid35271311, year = {2022}, author = {Cho, NH and Cheveralls, KC and Brunner, AD and Kim, K and Michaelis, AC and Raghavan, P and Kobayashi, H and Savy, L and Li, JY and Canaj, H and Kim, JYS and Stewart, EM and Gnann, C and McCarthy, F and Cabrera, JP and Brunetti, RM and Chhun, BB and Dingle, G and Hein, MY and Huang, B and Mehta, SB and Weissman, JS and Gómez-Sjöberg, R and Itzhak, DN and Royer, LA and Mann, M and Leonetti, MD}, title = {OpenCell: Endogenous tagging for the cartography of human cellular organization.}, journal = {Science (New York, N.Y.)}, volume = {375}, number = {6585}, pages = {eabi6983}, doi = {10.1126/science.abi6983}, pmid = {35271311}, issn = {1095-9203}, support = {F31 HL143882/HL/NHLBI NIH HHS/United States ; R01 GM131641/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; }, mesh = {CRISPR-Cas Systems ; Cluster Analysis ; Datasets as Topic ; Fluorescent Dyes ; HEK293 Cells ; Humans ; Immunoprecipitation ; Machine Learning ; Mass Spectrometry ; Microscopy, Confocal ; *Protein Interaction Mapping ; Proteins/*metabolism ; Proteome/*metabolism ; Proteomics/*methods ; RNA-Binding Proteins/metabolism ; Spatial Analysis ; }, abstract = {Elucidating the wiring diagram of the human cell is a central goal of the postgenomic era. We combined genome engineering, confocal live-cell imaging, mass spectrometry, and data science to systematically map the localization and interactions of human proteins. Our approach provides a data-driven description of the molecular and spatial networks that organize the proteome. Unsupervised clustering of these networks delineates functional communities that facilitate biological discovery. We found that remarkably precise functional information can be derived from protein localization patterns, which often contain enough information to identify molecular interactions, and that RNA binding proteins form a specific subgroup defined by unique interaction and localization properties. Paired with a fully interactive website (opencell.czbiohub.org), our work constitutes a resource for the quantitative cartography of human cellular organization.}, }
@article {pmid35271248, year = {2022}, author = {Bernhards, CB and Liem, AT and Berk, KL and Roth, PA and Gibbons, HS and Lux, MW}, title = {Putative Phenotypically Neutral Genomic Insertion Points in Prokaryotes.}, journal = {ACS synthetic biology}, volume = {11}, number = {4}, pages = {1681-1685}, pmid = {35271248}, issn = {2161-5063}, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Genome ; Genomics ; Software ; }, abstract = {The barriers to effective genome editing in diverse prokaryotic organisms have been falling at an accelerated rate. As editing becomes easier in more organisms, quickly identifying genomic locations to insert new genetic functions without disrupting organism fitness becomes increasingly useful. When the insertion is noncoding DNA for applications such as information storage or barcoding, a neutral insertion point can be especially important. Here we describe an approach to identify putatively neutral insertion sites in prokaryotes. An algorithm (targetFinder) finds convergently transcribed genes with gap sizes within a specified range, and looks for annotations within the gaps. We report putative editing targets for 10 common synthetic biology chassis organisms, including coverage of available RNA-seq data, and provide software to apply to others. We further experimentally evaluate the neutrality of six identified targets in Escherichia coli through insertion of a DNA barcode. We anticipate this information and the accompanying tool will prove useful for synthetic biologists seeking neutral insertion points for genome editing.}, }
@article {pmid35270040, year = {2022}, author = {Oh, HJ and Chung, E and Kim, J and Kim, MJ and Kim, GA and Lee, SH and Ra, K and Eom, K and Park, S and Chae, JH and Kim, JS and Lee, BC}, title = {Generation of a Dystrophin Mutant in Dog by Nuclear Transfer Using CRISPR/Cas9-Mediated Somatic Cells: A Preliminary Study.}, journal = {International journal of molecular sciences}, volume = {23}, number = {5}, pages = {}, pmid = {35270040}, issn = {1422-0067}, support = {IBS-R021-D1//Institute for Basic Science/ ; #550-20200076//Nature Cell/ ; }, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Dogs ; *Dystrophin/genetics/metabolism ; Gene Editing ; Muscle, Skeletal/metabolism ; *Muscular Dystrophy, Duchenne/genetics/therapy ; Nuclear Transfer Techniques ; }, abstract = {Dystrophinopathy is caused by mutations in the dystrophin gene, which lead to progressive muscle degeneration, necrosis, and finally, death. Recently, golden retrievers have been suggested as a useful animal model for studying human dystrophinopathy, but the model has limitations due to difficulty in maintaining the genetic background using conventional breeding. In this study, we successfully generated a dystrophin mutant dog using the CRISPR/Cas9 system and somatic cell nuclear transfer. The dystrophin mutant dog displayed phenotypes such as elevated serum creatine kinase, dystrophin deficiency, skeletal muscle defects, an abnormal electrocardiogram, and avoidance of ambulation. These results indicate that donor cells with CRISPR/Cas9 for a specific gene combined with the somatic cell nuclear transfer technique can efficiently produce a dystrophin mutant dog, which will help in the successful development of gene therapy drugs for dogs and humans.}, }
@article {pmid35269691, year = {2022}, author = {Sato, M and Nakamura, S and Inada, E and Takabayashi, S}, title = {Recent Advances in the Production of Genome-Edited Rats.}, journal = {International journal of molecular sciences}, volume = {23}, number = {5}, pages = {}, pmid = {35269691}, issn = {1422-0067}, support = {24580411//Japan Society for the Promotion of Science/ ; 16H05049//Japan Society for the Promotion of Science/ ; 21K10165//Japan Society for the Promotion of Science/ ; 16K07087//Japan Society for the Promotion of Science/ ; }, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Female ; Gene Editing/methods ; Genome/genetics ; Humans ; *Nucleic Acids ; Rats ; Transcription Activator-Like Effector Nucleases/genetics ; Zinc Finger Nucleases/genetics ; }, abstract = {The rat is an important animal model for understanding gene function and developing human disease models. Knocking out a gene function in rats was difficult until recently, when a series of genome editing (GE) technologies, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the type II bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated Cas9 (CRISPR/Cas9) systems were successfully applied for gene modification (as exemplified by gene-specific knockout and knock-in) in the endogenous target genes of various organisms including rats. Owing to its simple application for gene modification and its ease of use, the CRISPR/Cas9 system is now commonly used worldwide. The most important aspect of this process is the selection of the method used to deliver GE components to rat embryos. In earlier stages, the microinjection (MI) of GE components into the cytoplasm and/or nuclei of a zygote was frequently employed. However, this method is associated with the use of an expensive manipulator system, the skills required to operate it, and the egg transfer (ET) of MI-treated embryos to recipient females for further development. In vitro electroporation (EP) of zygotes is next recognized as a simple and rapid method to introduce GE components to produce GE animals. Furthermore, in vitro transduction of rat embryos with adeno-associated viruses is potentially effective for obtaining GE rats. However, these two approaches also require ET. The use of gene-engineered embryonic stem cells or spermatogonial stem cells appears to be of interest to obtain GE rats; however, the procedure itself is difficult and laborious. Genome-editing via oviductal nucleic acids delivery (GONAD) (or improved GONAD (i-GONAD)) is a novel method allowing for the in situ production of GE zygotes existing within the oviductal lumen. This can be performed by the simple intraoviductal injection of GE components and subsequent in vivo EP toward the injected oviducts and does not require ET. In this review, we describe the development of various approaches for producing GE rats together with an assessment of their technical advantages and limitations, and present new GE-related technologies and current achievements using those rats in relation to human diseases.}, }
@article {pmid35269602, year = {2022}, author = {Song, H and Ahn, JY and Yan, F and Ran, Y and Koo, O and Lee, GJ}, title = {Genetic Dissection of CRISPR-Cas9 Mediated Inheritance of Independently Targeted Alleles in Tobacco α-1,3-Fucosyltransferase 1 and β-1,2-Xylosyltransferase 1 Loci.}, journal = {International journal of molecular sciences}, volume = {23}, number = {5}, pages = {}, pmid = {35269602}, issn = {1422-0067}, mesh = {Alleles ; *CRISPR-Cas Systems/genetics ; Fucosyltransferases ; Gene Editing/methods ; Genes, Plant ; Humans ; Mutation ; Pentosyltransferases ; Plants, Genetically Modified/genetics ; *Tobacco/genetics ; }, abstract = {We determined the specificity of mutations induced by the CRISPR-Cas9 gene-editing system in tobacco (Nicotiana benthamiana) alleles and subsequent genetic stability. For this, we prepared 248 mutant plants using an Agrobacterium-delivered CRISPR-Cas9 system targeting α-1,3-fucosyltransferase 1 (FucT1) and β-1,2-xylosyltransferase1 (XylT1) genes, for which the mutation rates were 22.5% and 25%, respectively, with 20.5% for both loci. Individuals with wild-type (WT) alleles at the NbFucT1 locus in T0 were further segregated into chimeric progeny (37-54%) in the next generation, whereas homozygous T0 mutants tended to produce more (~70%) homozygotes than other bi-allelic and chimeric progenies in the T1 generation. Approximately 81.8% and 77.4% of the homozygous and bi-allelic mutations in T0 generation, respectively, were stably inherited in the next generation, and approximately 50% of the Cas9-free mutants were segregated in T2 generation. One homozygous mutant (Ta 161-1) with a +1 bp insertion in NbFucT1 and a -4 bp deletion in NbXylT1 was found to produce T2 progenies with the same alleles, indicating no activity of the integrated Cas9 irrespective of the insertion or deletion type. Our results provide empirical evidence regarding the genetic inheritance of alleles at CRISPR-targeted loci in tobacco transformants and indicate the potential factors contributing to further mutagenesis.}, }
@article {pmid35269584, year = {2022}, author = {Koo, CZ and Matthews, AL and Harrison, N and Szyroka, J and Nieswandt, B and Gardiner, EE and Poulter, NS and Tomlinson, MG}, title = {The Platelet Collagen Receptor GPVI Is Cleaved by Tspan15/ADAM10 and Tspan33/ADAM10 Molecular Scissors.}, journal = {International journal of molecular sciences}, volume = {23}, number = {5}, pages = {}, pmid = {35269584}, issn = {1422-0067}, support = {BB/P00783X/1 and MIBTP PhD Studentships/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; FS/18/9/33388/BHF_/British Heart Foundation/United Kingdom ; }, mesh = {ADAM10 Protein/*metabolism ; Amyloid Precursor Protein Secretases/*metabolism ; Blood Platelets/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Membrane Proteins/*metabolism ; Platelet Membrane Glycoproteins/*genetics/metabolism ; Protein Domains ; Proteolysis ; Substrate Specificity ; Tetraspanins/chemistry/*genetics/*metabolism ; }, abstract = {The platelet-activating collagen receptor GPVI represents the focus of clinical trials as an antiplatelet target for arterial thrombosis, and soluble GPVI is a plasma biomarker for several human diseases. A disintegrin and metalloproteinase 10 (ADAM10) acts as a 'molecular scissor' that cleaves the extracellular region from GPVI and many other substrates. ADAM10 interacts with six regulatory tetraspanin membrane proteins, Tspan5, Tspan10, Tspan14, Tspan15, Tspan17 and Tspan33, which are collectively termed the TspanC8s. These are emerging as regulators of ADAM10 substrate specificity. Human platelets express Tspan14, Tspan15 and Tspan33, but which of these regulates GPVI cleavage remains unknown. To address this, CRISPR/Cas9 knockout human cell lines were generated to show that Tspan15 and Tspan33 enact compensatory roles in GPVI cleavage, with Tspan15 bearing the more important role. To investigate this mechanism, a series of Tspan15 and GPVI mutant expression constructs were designed. The Tspan15 extracellular region was found to be critical in promoting GPVI cleavage, and appeared to achieve this by enabling ADAM10 to access the cleavage site at a particular distance above the membrane. These findings bear implications for the regulation of cleavage of other ADAM10 substrates, and provide new insights into post-translational regulation of the clinically relevant GPVI protein.}, }
@article {pmid35269459, year = {2022}, author = {Freudenberg, RA and Wittemeier, L and Einhaus, A and Baier, T and Kruse, O}, title = {The Spermidine Synthase Gene SPD1: A Novel Auxotrophic Marker for Chlamydomonas reinhardtii Designed by Enhanced CRISPR/Cas9 Gene Editing.}, journal = {Cells}, volume = {11}, number = {5}, pages = {}, pmid = {35269459}, issn = {2073-4409}, support = {34.EFRE-0300095/1703FI04//European Regional Development Fund/ ; }, mesh = {CRISPR-Cas Systems/genetics ; *Chlamydomonas reinhardtii/genetics/metabolism ; *Gene Editing/methods ; Spermidine/metabolism ; Spermidine Synthase/genetics/metabolism ; }, abstract = {Biotechnological application of the green microalga Chlamydomonas reinhardtii hinges on the availability of selectable markers for effective expression of multiple transgenes. However, biological safety concerns limit the establishment of new antibiotic resistance genes and until today, only a few auxotrophic markers exist for C. reinhardtii. The recent improvements in gene editing via CRISPR/Cas allow directed exploration of new endogenous selectable markers. Since editing frequencies remain comparably low, a Cas9-sgRNA ribonucleoprotein (RNP) delivery protocol was strategically optimized by applying nitrogen starvation to the pre-culture, which improved successful gene edits from 10% to 66% after pre-selection. Probing the essential polyamine biosynthesis pathway, the spermidine synthase gene (SPD1) is shown to be a potent selectable marker with versatile biotechnological applicability. Very low levels of spermidine (0.75 mg/L) were required to maintain normal mixotrophic and phototrophic growth in newly designed spermidine auxotrophic strains. Complementation of these strains with a synthetic SPD1 gene was achieved when the mature protein was expressed in the cytosol or targeted to the chloroplast. This work highlights the potential of new selectable markers for biotechnology as well as basic research and proposes an effective pipeline for the identification of new auxotrophies in C. reinhardtii.}, }
@article {pmid35269429, year = {2022}, author = {Parain, K and Lourdel, S and Donval, A and Chesneau, A and Borday, C and Bronchain, O and Locker, M and Perron, M}, title = {CRISPR/Cas9-Mediated Models of Retinitis Pigmentosa Reveal Differential Proliferative Response of Müller Cells between Xenopus laevis and Xenopus tropicalis.}, journal = {Cells}, volume = {11}, number = {5}, pages = {}, pmid = {35269429}, issn = {2073-4409}, support = {//Fondation pour la Recherche Médicale/ ; //Retina France/ ; //Fondation Valentin Haüy/ ; //UNADEV-ITMO-AVIESAN/ ; }, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Ependymoglial Cells/metabolism ; Retinal Rod Photoreceptor Cells/metabolism ; *Retinitis Pigmentosa/metabolism ; *Rhodopsin/genetics/metabolism ; Xenopus laevis/genetics/metabolism ; }, abstract = {Retinitis pigmentosa is an inherited retinal dystrophy that ultimately leads to blindness due to the progressive degeneration of rod photoreceptors and the subsequent non-cell autonomous death of cones. Rhodopsin is the most frequently mutated gene in this disease. We here developed rhodopsin gene editing-based models of retinitis pigmentosa in two Xenopus species, Xenopus laevis and Xenopus tropicalis, by using CRISPR/Cas9 technology. In both of them, loss of rhodopsin function results in massive rod cell degeneration characterized by progressive shortening of outer segments and occasional cell death. This is followed by cone morphology deterioration. Despite these apparently similar degenerative environments, we found that Müller glial cells behave differently in Xenopus laevis and Xenopus tropicalis. While a significant proportion of Müller cells re-enter into the cell cycle in Xenopus laevis, their proliferation remains extremely limited in Xenopus tropicalis. This work thus reveals divergent responses to retinal injury in closely related species. These models should help in the future to deepen our understanding of the mechanisms that have shaped regeneration during evolution, with tremendous differences across vertebrates.}, }
@article {pmid35266687, year = {2022}, author = {Mahas, A and Wang, Q and Marsic, T and Mahfouz, MM}, title = {Development of Cas12a-Based Cell-Free Small-Molecule Biosensors via Allosteric Regulation of CRISPR Array Expression.}, journal = {Analytical chemistry}, volume = {94}, number = {11}, pages = {4617-4626}, pmid = {35266687}, issn = {1520-6882}, mesh = {Allosteric Regulation ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA, Single-Stranded ; }, abstract = {Cell-free biosensors can detect various molecules, thus promising to transform the landscape of diagnostics. Here, we developed a simple, rapid, sensitive, and field-deployable small-molecule detection platform based on allosteric transcription factor (aTF)-regulated expression of a clustered regularly interspaced short palindromic repeats (CRISPR) array coupled to Cas12a activity. To this end, we engineered an expression cassette harboring a T7 promoter, an aTF binding sequence, a Cas12a CRISPR array, and protospacer adjacent motif-flanked Cas12a target sequences. In the presence of the ligand, dissociation of the aTF allows transcription of the CRISPR array; this leads to activation of Cas12a collateral activity, which cleaves a single-stranded DNA linker to free a quenched fluorophore, resulting in a rapid, significant increase of fluorescence. As a proof of concept, we used TetR as the aTF to detect different tetracycline antibiotics with high sensitivity and specificity and a simple, hand-held visualizer to develop a fluorescence-based visual readout. We also adapted a mobile phone application to further simplify the interpretation of the results. Finally, we showed that the reagents could be lyophilized to facilitate storage and distribution. This detection platform represents a valuable addition to the toolbox of cell-free, CRISPR-based biosensors, with great potential for in-field deployment to detect non-nucleic acid small molecules.}, }
@article {pmid35265984, year = {2022}, author = {Cremanns, M and Lange, F and Gatermann, SG and Pfennigwerth, N}, title = {Effect of sigma E on carbapenem resistance in OXA-48-producing Klebsiella pneumoniae.}, journal = {The Journal of antimicrobial chemotherapy}, volume = {}, number = {}, pages = {}, doi = {10.1093/jac/dkac078}, pmid = {35265984}, issn = {1460-2091}, support = {//Robert Koch Institute/ ; 1369-402//German Ministry of Health/ ; }, abstract = {OBJECTIVES: Resistance levels of Gram-negative bacteria producing OXA-48 carbapenemase can vary greatly and some of them can even be categorized as susceptible to imipenem and meropenem according to EUCAST breakpoints. This study aimed to reveal resistance mechanisms leading to varying levels of resistance to carbapenems in Klebsiella pneumoniae with blaOXA-48 submitted to the German National Reference Centre for MDR Gram-negative bacteria.<br><br>METHODS: Meropenem-susceptible clinical blaOXA-48-bearing K. pneumoniae isolates were put under gradually increasing selective pressure of meropenem. Clinical isolates and spontaneous meropenem-resistant mutants were whole-genome sequenced with Illumina and Oxford Nanopore Technology. Identified mutations apart from porin mutations were genetically constructed in the original clinical isolates using CRISPR/Cas. Clinical isolates and mutants were analysed for MICs, growth rates and expression of porins on mRNA and protein levels.<br><br>RESULTS: Mutations associated with meropenem resistance were predominantly found in ompK36, but in some cases ompK36 was unaffected. In two mutants, ISs within the rpoE (sigma factor E; σE) operon were detected, directly in or upstream of rseA. These IS1R elements were then inserted into the same position of the susceptible clinical isolates using CRISPR/Cas. CRISPR-rseA-rseB-rseC mutants showed higher resistance levels to carbapenems and cephalosporins, reduced growth rates and reduced expression of major porins OmpK36 and OmpK35 in quantitative RT-PCR and SDS-PAGE.<br><br>CONCLUSIONS: Enhanced synthesis of σE leads to increased resistance to cephalosporins and carbapenems in clinical K. pneumoniae isolates. This effect could be based upon remodelling of expression patterns of outer membrane proteins. The up-regulated σE stress response also leads to a significant reduction in growth rates.}, }
@article {pmid35265606, year = {2022}, author = {Shi, Y and Kang, L and Mu, R and Xu, M and Duan, X and Li, Y and Yang, C and Ding, JW and Wang, Q and Li, S}, title = {CRISPR/Cas12a-Enhanced Loop-Mediated Isothermal Amplification for the Visual Detection of Shigella flexneri.}, journal = {Frontiers in bioengineering and biotechnology}, volume = {10}, number = {}, pages = {845688}, pmid = {35265606}, issn = {2296-4185}, abstract = {Shigella flexneri is a serious threat to global public health, and a rapid detection method is urgently needed. The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system is widely used in gene editing, gene therapy, and in vitro diagnosis. Here, we combined loop-mediated isothermal amplification (LAMP) and CRISPR/Cas12a to develop a novel diagnostic test (CRISPR/Cas12a-E-LAMP) for the diagnosis of S. flexneri. The CRISPR/Cas12a-E-LAMP protocol conducts LAMP reaction for S. flexneri templates followed by CRISPR/Cas12a detection of predefined target sequences. LAMP primers and sgRNAs were designed to the highly conserved gene hypothetical protein (accession: AE014073, region: 4170556-4171,068) of S. flexneri. After the LAMP reaction at 60°C for 20 min, the pre-loaded CRISPR/Cas12a regents were mixed with the LAMP products in one tube at 37°C for 20 min, and the final results can be viewed by naked eyes with a total time of 40 min. The sensitivity of CRISPR/Cas12a-E-LAMP to detect S. flexneri was 4 × 100 copies/μl plasmids and without cross-reaction with other six closely related non-S. flexneri. Therefore, the CRISPR/Cas12a-E-LAMP assay is a useful method for the reliable and quick diagnosis of S. flexneri and may be applied in other pathogen infection detection.}, }
@article {pmid35264807, year = {2022}, author = {Ledford, H}, title = {Major CRISPR patent decision won't end tangled dispute.}, journal = {Nature}, volume = {603}, number = {7901}, pages = {373-374}, doi = {10.1038/d41586-022-00629-y}, pmid = {35264807}, issn = {1476-4687}, mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Dissent and Disputes ; Gene Editing ; }, }
@article {pmid35264732, year = {2022}, author = {}, title = {Fine-tuning epigenome editors.}, journal = {Nature biotechnology}, volume = {40}, number = {3}, pages = {281}, doi = {10.1038/s41587-022-01270-w}, pmid = {35264732}, issn = {1546-1696}, mesh = {CRISPR-Cas Systems ; *Epigenome ; *Gene Editing ; }, }
@article {pmid35264460, year = {2022}, author = {Christian, ML and Dapp, MJ and Scharffenberger, SC and Jones, H and Song, C and Frenkel, LM and Krumm, A and Mullins, JI and Rawlings, DJ}, title = {CRISPR/Cas9-Mediated Insertion of HIV Long Terminal Repeat within BACH2 Promotes Expansion of T Regulatory-like Cells.}, journal = {Journal of immunology (Baltimore, Md. : 1950)}, volume = {208}, number = {7}, pages = {1700-1710}, pmid = {35264460}, issn = {1550-6606}, support = {R01 DA040532/DA/NIDA NIH HHS/United States ; R61 DA047010/DA/NIDA NIH HHS/United States ; R01 CA206466/CA/NCI NIH HHS/United States ; R33 AI122361/AI/NIAID NIH HHS/United States ; R01 AI134419/AI/NIAID NIH HHS/United States ; R21 AI122361/AI/NIAID NIH HHS/United States ; P30 AI027757/AI/NIAID NIH HHS/United States ; R01 AI125026/AI/NIAID NIH HHS/United States ; }, mesh = {Basic-Leucine Zipper Transcription Factors/genetics ; *CRISPR-Cas Systems ; HIV Long Terminal Repeat/genetics ; *HIV-1/genetics ; Humans ; Virus Integration ; }, abstract = {One key barrier to curative therapies for HIV is the limited understanding of HIV persistence. HIV provirus integration sites (ISs) within BACH2 are common, and almost all sites mapped to date are located upstream of the start codon in the same transcriptional orientation as the gene. These unique features suggest the possibility of insertional mutagenesis at this location. Using CRISPR/Cas9-based homology-directed repair in primary human CD4+ T cells, we directly modeled the effects of HIV integration within BACH2 Integration of the HIV long terminal repeat (LTR) and major splice donor increased BACH2 mRNA and protein levels, altered gene expression, and promoted selective outgrowth of an activated, proliferative, and T regulatory-like cell population. In contrast, introduction of the HIV-LTR alone or an HIV-LTR-major splice donor construct into STAT5B, a second common HIV IS, had no functional impact. Thus, HIV LTR-driven BACH2 expression modulates T cell programming and leads to cellular outgrowth and unique phenotypic changes, findings that support a direct role for IS-dependent HIV-1 persistence.}, }
@article {pmid35263584, year = {2022}, author = {Tian, S and Liu, Y and Appleton, E and Wang, H and Church, GM and Dong, M}, title = {Targeted intracellular delivery of Cas13 and Cas9 nucleases using bacterial toxin-based platforms.}, journal = {Cell reports}, volume = {38}, number = {10}, pages = {110476}, pmid = {35263584}, issn = {2211-1247}, support = {R21 NS106159/NS/NINDS NIH HHS/United States ; R01 NS080833/NS/NINDS NIH HHS/United States ; R01 AI132387/AI/NIAID NIH HHS/United States ; R01 AI139087/AI/NIAID NIH HHS/United States ; R01 NS117626/NS/NINDS NIH HHS/United States ; P30 HD018655/HD/NICHD NIH HHS/United States ; P30 DK034854/DK/NIDDK NIH HHS/United States ; }, mesh = {*Bacterial Toxins/genetics ; *COVID-19 ; CRISPR-Cas Systems ; Gene Editing ; Humans ; RNA, Guide/metabolism ; SARS-CoV-2 ; }, abstract = {Targeted delivery of therapeutic proteins toward specific cells and across cell membranes remains major challenges. Here, we develop protein-based delivery systems utilizing detoxified single-chain bacterial toxins such as diphtheria toxin (DT) and botulinum neurotoxin (BoNT)-like toxin, BoNT/X, as carriers. The system can deliver large protein cargoes including Cas13a, CasRx, Cas9, and Cre recombinase into cells in a receptor-dependent manner, although delivery of ribonucleoproteins containing guide RNAs is not successful. Delivery of Cas13a and CasRx, together with guide RNA expression, reduces mRNAs encoding GFP, SARS-CoV-2 fragments, and endogenous proteins PPIB, KRAS, and CXCR4 in multiple cell lines. Delivery of Cre recombinase modifies the reporter loci in cells. Delivery of Cas9, together with guide RNA expression, generates mutations at the targeted genomic sites in cell lines and induced pluripotent stem cell (iPSC)-derived human neurons. These findings establish modular delivery systems based on single-chain bacterial toxins for delivery of membrane-impermeable therapeutics into targeted cells.}, }
@article {pmid35263578, year = {2022}, author = {Michl, J and Wang, Y and Monterisi, S and Blaszczak, W and Beveridge, R and Bridges, EM and Koth, J and Bodmer, WF and Swietach, P}, title = {CRISPR-Cas9 screen identifies oxidative phosphorylation as essential for cancer cell survival at low extracellular pH.}, journal = {Cell reports}, volume = {38}, number = {10}, pages = {110493}, pmid = {35263578}, issn = {2211-1247}, support = {/WT_/Wellcome Trust/United Kingdom ; }, mesh = {CRISPR-Cas Systems/genetics ; Cell Survival/genetics ; Humans ; Hydrogen-Ion Concentration ; *Neoplasms/genetics ; *Oxidative Phosphorylation ; }, abstract = {Unlike most cell types, many cancer cells survive at low extracellular pH (pHe), a chemical signature of tumors. Genes that facilitate survival under acid stress are therefore potential targets for cancer therapies. We performed a genome-wide CRISPR-Cas9 cell viability screen at physiological and acidic conditions to systematically identify gene knockouts associated with pH-related fitness defects in colorectal cancer cells. Knockouts of genes involved in oxidative phosphorylation (NDUFS1) and iron-sulfur cluster biogenesis (IBA57, NFU1) grew well at physiological pHe, but underwent profound cell death under acidic conditions. We identified several small-molecule inhibitors of mitochondrial metabolism that can kill cancer cells at low pHe only. Xenografts established from NDUFS1-/- cells grew considerably slower than their wild-type controls, but growth could be stimulated with systemic bicarbonate therapy that lessens the tumoral acid stress. These findings raise the possibility of therapeutically targeting mitochondrial metabolism in combination with acid stress as a cancer treatment option.}, }
@article {pmid35263090, year = {2022}, author = {Kang, Y and Su, G and Yu, Y and Cao, J and Wang, J and Yan, B}, title = {CRISPR-Cas12a-Based Aptasensor for On-Site and Highly Sensitive Detection of Microcystin-LR in Freshwater.}, journal = {Environmental science &amp; technology}, volume = {56}, number = {7}, pages = {4101-4110}, doi = {10.1021/acs.est.1c06733}, pmid = {35263090}, issn = {1520-5851}, mesh = {*Aptamers, Nucleotide/metabolism ; *Biosensing Techniques/methods ; CRISPR-Cas Systems ; Fresh Water ; Limit of Detection ; Marine Toxins ; Microcystins/metabolism ; }, abstract = {On-site monitoring of trace organic pollutants with facile methods is critical to environmental pollutant prevention and control. Herein, we proposed a CRISPR-Cas12a-based aptasensor platform (named as MC-LR-Casor) for on-site and sensitive detection of microcystin-LR (MC-LR). After hybridization with blocker DNA, the MC-LR aptamers were conjugated to magnetic beads (MBs) to get the MB aptasensor. In the presence of MC-LR, their interactions with aptamers were triggered and the specific binding caused the release of blocker DNA. Using the programmability of the CRISPR-Cas system, the released blocker DNA was designed to activate a Cas12a-crRNA complex. Single strand DNA reporters were rapidly cleaved by the complex. Signal readout could be achieved by fluorometer or lateral flow strips, which were positively correlated to MC-LR concentration. Benefiting from the CRISPR-Cas12a amplification system, the proposed sensing platform exhibited high sensitivity and reached the limit of detection of ∼3 × 10-6 μg/L (fluorescence method) or 1 × 10-3 μg/L (lateral flow assay). In addition, the MC-LR-Casor showed excellent selectivity and good recovery rates, demonstrating their good applicability for real water sample analysis. During the whole assay, only two steps of incubation at a constant temperature were required and the results could be visualized when employing flow strips. Therefore, the proposed assay offered a simple and convenient alternative for in situ MC-LR monitoring, which may hold great promise for future environmental surveillance.}, }
@article {pmid35261159, year = {2022}, author = {Saw, PE and Cui, GH and Xu, X}, title = {Nanoparticles-Mediated CRISPR/Cas Gene Editing Delivery System.}, journal = {ChemMedChem}, volume = {17}, number = {9}, pages = {e202100777}, doi = {10.1002/cmdc.202100777}, pmid = {35261159}, issn = {1860-7187}, support = {82050410363//National Natural Science Foundation of China/ ; 82072930//National Natural Science Foundation of China/ ; 2018A050506033//International Scientific and Technological Department of Guangdong Province/ ; 2019020015//Guangzhou Science and Technology Bureau/ ; }, mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing ; Genetic Therapy ; *Nanoparticles ; }, abstract = {CRISPR/Cas system has become one of the most powerful techologies in biomedical research, and has showed great potentials in the gene related diseases. However, efficient delivery systems of CRISPR/Cas to target cells remains challenging. In recent years, nanoparticles have showned great potentials for the delivery of CRISPR/Cas systems. This paper mainly approaches the development and new strategies of CRISPR/Cas delivery systems, as well as their application in the clinical diseases. By summarizing the CRISPR/Cas systems delivery, new strategies are expected for the gene therapy.}, }
@article {pmid35260779, year = {2022}, author = {Nakagawa, R and Ishiguro, S and Okazaki, S and Mori, H and Tanaka, M and Aburatani, H and Yachie, N and Nishimasu, H and Nureki, O}, title = {Engineered Campylobacter jejuni Cas9 variant with enhanced activity and broader targeting range.}, journal = {Communications biology}, volume = {5}, number = {1}, pages = {211}, pmid = {35260779}, issn = {2399-3642}, support = {JP19am0401005//Japan Agency for Medical Research and Development (AMED)/ ; }, mesh = {*CRISPR-Cas Systems ; *Campylobacter jejuni/genetics ; Gene Editing ; Humans ; }, abstract = {The RNA-guided DNA endonuclease Cas9 is a versatile genome-editing tool. However, the molecular weight of the commonly used Streptococcus pyogenes Cas9 is relatively large. Consequently, its gene cannot be efficiently packaged into an adeno-associated virus vector, thereby limiting its applications for therapeutic genome editing. Here, we biochemically characterized the compact Cas9 from Campylobacter jejuni (CjCas9) and found that CjCas9 has a previously unrecognized preference for the N3VRYAC protospacer adjacent motif. We thus rationally engineered a CjCas9 variant (enCjCas9), which exhibits enhanced cleavage activity and a broader targeting range both in vitro and in human cells, as compared with CjCas9. Furthermore, a nickase version of enCjCas9, but not CjCas9, fused with a cytosine deaminase mediated C-to-T conversions in human cells. Overall, our findings expand the CRISPR-Cas toolbox for therapeutic genome engineering.}, }
@article {pmid35260651, year = {2022}, author = {Pini, V and Mariot, V and Dumonceaux, J and Counsell, J and O'Neill, HC and Farmer, S and Conti, F and Muntoni, F}, title = {Transiently expressed CRISPR/Cas9 induces wild-type dystrophin in vitro in DMD patient myoblasts carrying duplications.}, journal = {Scientific reports}, volume = {12}, number = {1}, pages = {3756}, pmid = {35260651}, issn = {2045-2322}, support = {/MRC_/Medical Research Council/United Kingdom ; /DH_/Department of Health/United Kingdom ; }, mesh = {*CRISPR-Cas Systems/genetics ; Dystrophin/genetics/metabolism ; Endonucleases/genetics ; Gene Editing/methods ; Genetic Therapy/methods ; Humans ; *Muscular Dystrophy, Duchenne/genetics/metabolism/therapy ; Myoblasts/metabolism ; }, abstract = {Among the mutations arising in the DMD gene and causing Duchenne Muscular Dystrophy (DMD), 10-15% are multi-exon duplications. There are no current therapeutic approaches with the ability to excise large multi-exon duplications, leaving this patient cohort without mutation-specific treatment. Using CRISPR/Cas9 could provide a valid alternative to achieve targeted excision of genomic duplications of any size. Here we show that the expression of a single CRISPR/Cas9 nuclease targeting a genomic region within a DMD duplication can restore the production of wild-type dystrophin in vitro. We assessed the extent of dystrophin repair following both constitutive and transient nuclease expression by either transducing DMD patient-derived myoblasts with integrating lentiviral vectors or electroporating them with CRISPR/Cas9 expressing plasmids. Comparing genomic, transcript and protein data, we observed that both continuous and transient nuclease expression resulted in approximately 50% dystrophin protein restoration in treated myoblasts. Our data demonstrate that a high transient expression profile of Cas9 circumvents its requirement of continuous expression within the cell for targeting DMD duplications. This proof-of-concept study therefore helps progress towards a clinically relevant gene editing strategy for in vivo dystrophin restoration, by highlighting important considerations for optimizing future therapeutic approaches.}, }
@article {pmid35260581, year = {2022}, author = {Yin, J and Lu, R and Xin, C and Wang, Y and Ling, X and Li, D and Zhang, W and Liu, M and Xie, W and Kong, L and Si, W and Wei, P and Xiao, B and Lee, HY and Liu, T and Hu, J}, title = {Cas9 exo-endonuclease eliminates chromosomal translocations during genome editing.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {1204}, pmid = {35260581}, issn = {2041-1723}, mesh = {*CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; Endonucleases/genetics/metabolism ; *Gene Editing ; Humans ; RNA, Guide/chemistry/genetics ; Translocation, Genetic ; }, abstract = {The mechanism underlying unwanted structural variations induced by CRISPR-Cas9 remains poorly understood, and no effective strategy is available to inhibit the generation of these byproducts. Here we find that the generation of a high level of translocations is dependent on repeated cleavage at the Cas9-targeting sites. Therefore, we employ a strategy in which Cas9 is fused with optimized TREX2 to generate Cas9TX, a Cas9 exo-endonuclease, which prevents perfect DNA repair and thereby avoids repeated cleavage. In comparison with CRISPR-Cas9, CRISPR-Cas9TX greatly suppressed translocation levels and enhanced the editing efficiency of single-site editing. The number of large deletions associated with Cas9TX was also reduced to very low level. The application of CRISPR-Cas9TX for multiplex gene editing in chimeric antigen receptor T cells nearly eliminated deleterious chromosomal translocations. We report the mechanism underlying translocations induced by Cas9, and propose a general strategy for reducing chromosomal abnormalities induced by CRISPR-RNA-guided endonucleases.}, }
@article {pmid35258835, year = {2022}, author = {Decaestecker, W and Bollier, N and Buono, RA and Nowack, MK and Jacobs, TB}, title = {Protoplast Preparation and Fluorescence-Activated Cell Sorting for the Evaluation of Targeted Mutagenesis in Plant Cells.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2464}, number = {}, pages = {205-221}, pmid = {35258835}, issn = {1940-6029}, mesh = {*Arabidopsis/genetics ; CRISPR-Cas Systems ; Flow Cytometry/methods ; Mutagenesis ; Plant Cells ; *Protoplasts ; }, abstract = {Fluorescence-activated cell sorting (FACS) allows for the enrichment of specific plant cell populations after protoplasting. In this book chapter, we describe the transformation and protoplasting of an Arabidopsis thaliana cell suspension culture (PSB-D, derived from MM2d) that can be used for the evaluation of CRISPR vectors in a subpopulation of cells. We also describe the protoplasting of Arabidopsis thaliana cells from the roots and stomatal lineage for the evaluation of tissue-specific gene editing. These protocols allow us to rapidly and accurately quantify various CRISPR systems in plant cells.}, }
@article {pmid35258833, year = {2022}, author = {Patil, GB and Stupar, RM and Zhang, F}, title = {Protoplast Isolation, Transfection, and Gene Editing for Soybean (Glycine max).}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2464}, number = {}, pages = {173-186}, pmid = {35258833}, issn = {1940-6029}, mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Plant Breeding ; *Protoplasts/metabolism ; Ribonucleoproteins/metabolism ; Soybeans/genetics/metabolism ; Transfection ; }, abstract = {Protoplast is a versatile system for conducting cell-based assays, analyzing diverse signaling pathways, studying functions of cellular machineries, and functional genomics screening. Protoplast engineering has become an important tool for basic plant molecular biology research and developing genome-edited crops. This system allows the direct delivery of DNA, RNA, or proteins into plant cells and provides a high-throughput system to validate gene-editing reagents. It also facilitates the delivery of homology-directed repair templates (donor molecules) into plant cells, enabling precise DNA edits in the genome. There is a great deal of interest in the plant community to develop these precise edits, as they may expand the potential for developing value-added traits which may be difficult to achieve by other gene-editing applications and/or traditional breeding alone. This chapter provides improved working protocols for isolating and transforming protoplast from immature soybean seeds with 44% of transfection efficiency validated by the green fluorescent protein reporter. We also describe a method for gene editing in soybean protoplasts using single guide RNA molecules.}, }
@article {pmid35258832, year = {2022}, author = {Kang, M and Lee, K and Wang, K}, title = {Efficient Protoplast Isolation and DNA Transfection for Winter Oilseed Crops, Pennycress (Thlaspi arvense) and Camelina (Camelina sativa).}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2464}, number = {}, pages = {153-171}, pmid = {35258832}, issn = {1940-6029}, mesh = {Crops, Agricultural/genetics ; DNA/metabolism ; Protoplasts ; *Thlaspi/genetics/metabolism ; Transfection ; }, abstract = {Pennycress (Thlaspi arvense) and camelina (Camelina sativa) are nonfood winter oilseed crops that have the potential to contribute to sustainable biofuel production. However, undesired agronomic traits of pennycress and camelina currently hinder broad cultivation of these plants in the field. Recently, genome editing using the CRISPR-Cas technology has been applied to improve poor agronomic traits such as the weedy phenotype of pennycress and the oxidation susceptible lipid profile of camelina. In these works, the CRISPR reagents were introduced into the plants using the Agrobacterium-mediated floral dipping method. For accelerated domestication and value improvements of these winter oilseed crops, DNA-free genome editing platform and easy evaluation method of the CRISPR-Cas reagents are highly desirable. Cell wall-free protoplasts are great material to expand the use of gene engineering tools. In this chapter, we present a step-by-step guide to the mesophyll protoplast isolation from in vitro culture-grown pennycress and soil-grown camelina. The protocol also includes procedures for DNA transfection and protoplast viability test using fluorescein diacetate. With this protocol, we can isolate an average of 6 × 106 cells from pennycress and 3 × 106 cells from camelina per gram of fresh leaf tissues. Using a 7.3 kb plasmid DNA carrying green and red fluorescent protein marker genes, we can achieve an average transfection rate of 40% validated by flow cytometry for both plants.}, }
@article {pmid35258825, year = {2022}, author = {Banakar, R and Rai, KM and Zhang, F}, title = {CRISPR DNA- and RNP-Mediated Genome Editing via Nicotiana benthamiana Protoplast Transformation and Regeneration.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2464}, number = {}, pages = {65-82}, pmid = {35258825}, issn = {1940-6029}, mesh = {CRISPR-Cas Systems/genetics ; DNA ; *Gene Editing/methods ; Genome, Plant ; *Protoplasts ; Ribonucleoproteins/genetics ; Tobacco/genetics ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated system) has become the multipurpose tool to manipulate plant genome via their programmable sequence recognition, binding, and cleavage activities. Efficient plant genome modification often requires robust plant transformation. For most plant species, the CRISPR/Cas reagents are delivered into plants as plasmids by Agrobacterium-mediated T-DNA transfer or biolistic approaches. However, these methods are generally inefficient, heavily genotype dependent, and low throughput. Among the alternative plant transformation approaches, the protoplast-based transformation holds the potential to directly deliver DNA, RNA, or protein molecules into plant cells in an efficient and high-throughput manner. Here, we presented a robust and simplified protocol for protoplast-based DNA/ribonucleoprotein (RNP)-mediated genome editing in the model species Nicotiana benthamiana. Using this protocol, we have achieved the gene editing efficiency at 30-60% in protoplasts and 50-80% in regenerated calli and plants. The edited protoplasts can be readily regenerated without selection agents owing to highly efficient DNA or preassembled RNP transformation frequency. Lastly, this protocol utilized an improved culture media regime to overcome the complex media composition used in the previous studies. It offers quick turnaround time and higher throughput to facilitate the development of new genetic engineering technologies and holds the promise to combine with other genetic and genomic tools for fundamental and translational plant research.}, }
@article {pmid35258824, year = {2022}, author = {Wu, FH and Yuan, YH and Hsu, CT and Cheng, QW and Lin, CS}, title = {Application of Protoplast Regeneration to CRISPR/Cas9 Mutagenesis in Nicotiana tabacum.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2464}, number = {}, pages = {49-64}, pmid = {35258824}, issn = {1940-6029}, mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Mutagenesis ; *Protoplasts/metabolism ; Tobacco/genetics ; }, abstract = {Protoplast transfection is widely used in plant research to rapidly evaluate RNA degradation, reporter assay, gene expression, subcellular localization, and protein-protein interactions. In order to successfully use protoplast transfection with the newly emerging clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) protein editing platform, high yield of protoplasts, stable transfection efficiency, and reliable regeneration protocols are necessary. The Nicotiana tabacum transient protoplast transfection and regeneration system can effectively obtain target gene mutations in regenerated plants without transgenes and is thus a very attractive technique for evaluating gene editing reagents using CRISPR/Cas-based systems. Here, we describe in detail sterilized seed germination, culture conditions, isolation of Nicotiana tabacum protoplasts from tissue culture explants, construction of a vector containing the Cas protein and sgRNA cassette, highly efficient polyethylene glycol-calcium transient transfection of plasmids delivered into protoplasts, evaluation of mutagenesis efficiency and genotype analysis from protoplasts and regenerated plants, and the regeneration conditions to obtain CRISPR-edited plants from single protoplasts.}, }
@article {pmid35258601, year = {2022}, author = {Rybnicky, GA and Fackler, NA and Karim, AS and Köpke, M and Jewett, MC}, title = {Spacer2PAM: A computational framework to guide experimental determination of functional CRISPR-Cas system PAM sequences.}, journal = {Nucleic acids research}, volume = {50}, number = {6}, pages = {3523-3534}, pmid = {35258601}, issn = {1362-4962}, mesh = {*CRISPR-Cas Systems/genetics ; Clostridium/genetics ; Computational Biology/*methods ; Gene Library ; Nucleotide Motifs ; }, abstract = {RNA-guided nucleases from CRISPR-Cas systems expand opportunities for precise, targeted genome modification. Endogenous CRISPR-Cas systems in many prokaryotes are attractive to circumvent expression, functionality, and unintended activity hurdles posed by heterologous CRISPR-Cas effectors. However, each CRISPR-Cas system recognizes a unique set of protospacer adjacent motifs (PAMs), which requires identification by extensive screening of randomized DNA libraries. This challenge hinders development of endogenous CRISPR-Cas systems, especially those based on multi-protein effectors and in organisms that are slow-growing or have transformation idiosyncrasies. To address this challenge, we present Spacer2PAM, an easy-to-use, easy-to-interpret R package built to predict and guide experimental determination of functional PAM sequences for any CRISPR-Cas system given its corresponding CRISPR array as input. Spacer2PAM can be used in a 'Quick' method to generate a single PAM prediction or in a 'Comprehensive' method to inform targeted PAM libraries small enough to screen in difficult to transform organisms. We demonstrate Spacer2PAM by predicting PAM sequences for industrially relevant organisms and experimentally identifying seven PAM sequences that mediate interference from the Spacer2PAM-informed PAM library for the type I-B CRISPR-Cas system from Clostridium autoethanogenum. We anticipate that Spacer2PAM will facilitate the use of endogenous CRISPR-Cas systems for industrial biotechnology and synthetic biology.}, }
@article {pmid35257575, year = {2022}, author = {Zhang, D and Liu, L and Jin, S and Tota, E and Li, Z and Piao, X and Zhang, X and Fu, XD and Devaraj, NK}, title = {Site-Specific and Enzymatic Cross-Linking of sgRNA Enables Wavelength-Selectable Photoactivated Control of CRISPR Gene Editing.}, journal = {Journal of the American Chemical Society}, volume = {144}, number = {10}, pages = {4487-4495}, doi = {10.1021/jacs.1c12166}, pmid = {35257575}, issn = {1520-5126}, support = {R01 GM123285/GM/NIGMS NIH HHS/United States ; R35 GM141939/GM/NIGMS NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Mammals/genetics ; *RNA, Guide/genetics ; }, abstract = {Chemical cross-linking enables rapid identification of RNA-protein and RNA-nucleic acid inter- and intramolecular interactions. However, no method exists to site-specifically and covalently cross-link two user-defined sites within an RNA. Here, we develop RNA-CLAMP, which enables site-specific and enzymatic cross-linking (clamping) of two selected guanine residues within an RNA. Intramolecular clamping can disrupt normal RNA function, whereas subsequent photocleavage of the cross-linker restores activity. We used RNA-CLAMP to clamp two stem loops within the single-guide RNA (sgRNA) of the CRISPR-Cas9 gene editing system via a photocleavable cross-linker, completely inhibiting gene editing. Visible light irradiation cleaved the cross-linker and restored gene editing with high spatiotemporal resolution. Design of two photocleavable linkers responsive to different wavelengths of light allowed multiplexed photoactivation of gene editing in mammalian cells. This photoactivated CRISPR-Cas9 gene editing platform benefits from undetectable background activity, provides a choice of activation wavelengths, and has multiplexing capabilities.}, }
@article {pmid35255977, year = {2022}, author = {Li, L and Yi, H and Liu, Z and Long, P and Pan, T and Huang, Y and Li, Y and Li, Q and Ma, Y}, title = {Genetic correction of concurrent α- and β-thalassemia patient-derived pluripotent stem cells by the CRISPR-Cas9 technology.}, journal = {Stem cell research &amp; therapy}, volume = {13}, number = {1}, pages = {102}, pmid = {35255977}, issn = {1757-6512}, mesh = {CRISPR-Cas Systems/genetics ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Mutation ; Technology ; beta-Globins/genetics/metabolism ; *beta-Thalassemia/genetics/metabolism/therapy ; }, abstract = {BACKGROUND: Thalassemia is a genetic blood disorder characterized by decreased hemoglobin production. Severe anemia can damage organs and severe threat to life safety. Allogeneic transplantation of bone marrow-derived hematopoietic stem cell (HSCs) at present represents a promising therapeutic approach for thalassemia. However, immune rejection and lack of HLA-matched donors limited its clinical application. In recent years, human-induced pluripotent stem cells (hiPSCs) technology offers prospects for autologous cell-based therapy since it could avoid the immunological problems mentioned above.<br><br>METHODS: In the present study, we established a new hiPSCs line derived from amniotic cells of a fetus with a homozygous β41-42 (TCTT) deletion mutation in the HBB gene and a heterozygous Westmead mutation (C > G) in the HBA2 gene. We designed a CRISPR-Cas9 to target these casual mutations and corrected them. Gene-corrected off-target analysis was performed by whole-exome capture sequencing. The corrected hiPSCs were analyzed by teratoma formation and erythroblasts differentiation assays.<br><br>RESULTS: These mutations were corrected with linearized donor DNA through CRISPR/Cas9-mediated homology-directed repair. Corrections of hiPSCs were validated by sequences. The corrected hiPSCs retain normal pluripotency. Moreover, they could be differentiated into hematopoietic progenitors, which proves that they maintain the multilineage differentiation potential.<br><br>CONCLUSIONS: We designed sgRNAs and demonstrated that these sgRNAs facilitating the CRISPR-Cas9 genomic editing system could be applied to correct concurrent α- and β-thalassemia in patient-derived hiPSCs. In the future, these corrected hiPSCs can be applied for autologous transplantation in patients with concurrent α- and β-thalassemia.}, }
@article {pmid35254919, year = {2022}, author = {Zheng, Y and VanDusen, NJ and Butler, CE and Ma, Q and King, JS and Pu, WT}, title = {Efficient In Vivo Homology-Directed Repair Within Cardiomyocytes.}, journal = {Circulation}, volume = {145}, number = {10}, pages = {787-789}, doi = {10.1161/CIRCULATIONAHA.120.052383}, pmid = {35254919}, issn = {1524-4539}, support = {K99 HL143194/HL/NHLBI NIH HHS/United States ; R01 HL146634/HL/NHLBI NIH HHS/United States ; }, mesh = {CRISPR-Cas Systems ; Gene Editing ; Humans ; *Myocytes, Cardiac ; *Recombinational DNA Repair ; }, }
@article {pmid35254721, year = {2022}, author = {Grotz, S and Schäfer, J and Wunderlich, KA and Ellederova, Z and Auch, H and Bähr, A and Runa-Vochozkova, P and Fadl, J and Arnold, V and Ardan, T and Veith, M and Santamaria, G and Dhom, G and Hitzl, W and Kessler, B and Eckardt, C and Klein, J and Brymova, A and Linnert, J and Kurome, M and Zakharchenko, V and Fischer, A and Blutke, A and Döring, A and Suchankova, S and Popelar, J and Rodríguez-Bocanegra, E and Dlugaiczyk, J and Straka, H and May-Simera, H and Wang, W and Laugwitz, KL and Vandenberghe, LH and Wolf, E and Nagel-Wolfrum, K and Peters, T and Motlik, J and Fischer, MD and Wolfrum, U and Klymiuk, N}, title = {Early disruption of photoreceptor cell architecture and loss of vision in a humanized pig model of usher syndromes.}, journal = {EMBO molecular medicine}, volume = {14}, number = {4}, pages = {e14817}, pmid = {35254721}, issn = {1757-4684}, mesh = {Animals ; Cell Cycle Proteins/genetics ; Cytoskeletal Proteins ; Humans ; Photoreceptor Cells ; Swine ; *Usher Syndromes/genetics/metabolism/therapy ; }, abstract = {Usher syndrome (USH) is the most common form of monogenic deaf-blindness. Loss of vision is untreatable and there are no suitable animal models for testing therapeutic strategies of the ocular constituent of USH, so far. By introducing a human mutation into the harmonin-encoding USH1C gene in pigs, we generated the first translational animal model for USH type 1 with characteristic hearing defect, vestibular dysfunction, and visual impairment. Changes in photoreceptor architecture, quantitative motion analysis, and electroretinography were characteristics of the reduced retinal virtue in USH1C pigs. Fibroblasts from USH1C pigs or USH1C patients showed significantly elongated primary cilia, confirming USH as a true and general ciliopathy. Primary cells also proved their capacity for assessing the therapeutic potential of CRISPR/Cas-mediated gene repair or gene therapy in vitro. AAV-based delivery of harmonin into the eye of USH1C pigs indicated therapeutic efficacy in vivo.}, }
@article {pmid35254529, year = {2022}, author = {de Souza Moraes, T and van Es, SW and Hernández-Pinzón, I and Kirschner, GK and van der Wal, F and da Silveira, SR and Busscher-Lange, J and Angenent, GC and Moscou, M and Immink, RGH and van Esse, GW}, title = {The TCP transcription factor HvTB2 heterodimerizes with VRS5 and controls spike architecture in barley.}, journal = {Plant reproduction}, volume = {}, number = {}, pages = {}, pmid = {35254529}, issn = {2194-7961}, support = {VENI 15060//Dutch Research Council (NWO)/ ; }, abstract = {KEY MESSAGE: Understanding the molecular network, including protein-protein interactions, of VRS5 provide new routes towards the identification of other key regulators of plant architecture in barley. The TCP transcriptional regulator TEOSINTE BRANCHED 1 (TB1) is a key regulator of plant architecture. In barley, an important cereal crop, HvTB1 (also referred to as VULGARE SIX-ROWED spike (VRS) 5), inhibits the outgrowth of side shoots, or tillers, and grains. Despite its key role in barley development, there is limited knowledge on the molecular network that is utilized by VRS5. In this work, we performed protein-protein interaction studies of VRS5. Our analysis shows that VRS5 potentially interacts with a diverse set of proteins, including other class II TCP's, NF-Y TF, but also chromatin remodelers. Zooming in on the interaction capacity of VRS5 with other TCP TFs shows that VRS5 preferably interacts with other class II TCP TFs in the TB1 clade. Induced mutagenesis through CRISPR-Cas of one of the putative VRS5 interactors, HvTB2 (also referred to as COMPOSITUM 1 and BRANCHED AND INDETERMINATE SPIKELET 1), resulted in plants that have lost their characteristic unbranched spike architecture. More specifically, hvtb2 mutants exhibited branches arising at the main spike, suggesting that HvTB2 acts as inhibitor of branching. Our protein-protein interaction studies of VRS5 resulted in the identification of HvTB2 as putative interactor of VRS5, another key regulator of spike architecture in barley. The study presented here provides a first step to underpin the protein-protein interactome of VRS5 and to identify other, yet unknown, key regulators of barley plant architecture.}, }
@article {pmid35252043, year = {2022}, author = {Yin, D and Yin, L and Wang, J and Shen, X and Pan, X and Hou, H and Zhao, R and Hu, X and Wang, G and Qi, K and Dai, Y}, title = {Visual Detection of Duck Tembusu Virus With CRISPR/Cas13: A Sensitive and Specific Point-of-Care Detection.}, journal = {Frontiers in cellular and infection microbiology}, volume = {12}, number = {}, pages = {848365}, pmid = {35252043}, issn = {2235-2988}, mesh = {Animals ; CRISPR-Cas Systems ; Flavivirus ; *Flavivirus Infections/diagnosis/veterinary ; Point-of-Care Systems ; *Poultry Diseases/diagnosis ; Sensitivity and Specificity ; }, abstract = {Duck tembusu virus (DTMUV), which causes huge economic losses for the poultry industries in Southeast Asia and China, was first identified in 2010. DTMUV disease has become an important disease that endangers the duck industry. A sensitive, accurate, and convenient DTMUV detection method is an important means to reduce the occurrence of the disease. In this study, a CRISPR/Cas13a system was combined with recombinase polymerase amplification to develop a convenient diagnostic method to detect DTMUV. The novel method was based on isothermal detection at 37°C, and the detection was used for visual readout or real-time analysis. The assay was highly sensitive and specific, with a detection limit of 1 copy/μL of the target gene and showed no cross-reactivity with other pathogens. The enhanced Cas13a detection worked well with clinical samples. Overall, a visual, sensitive, and specific nucleic acid detection method based on CRISPR/Cas13a proved to be a powerful tool for detecting DTMUV.}, }
@article {pmid35252020, year = {2021}, author = {Sharma, R and Avendaño Rangel, F and Reis-Cunha, JL and Marques, LP and Figueira, CP and Borba, PB and Viana, SM and Beneke, T and Bartholomeu, DC and de Oliveira, CI}, title = {Targeted Deletion of Centrin in Leishmania braziliensis Using CRISPR-Cas9-Based Editing.}, journal = {Frontiers in cellular and infection microbiology}, volume = {11}, number = {}, pages = {790418}, pmid = {35252020}, issn = {2235-2988}, mesh = {Animals ; CRISPR-Cas Systems ; *Leishmania ; *Leishmania braziliensis/genetics ; *Leishmaniasis, Cutaneous/parasitology ; Mice ; Mice, Inbred BALB C ; Trimethoprim, Sulfamethoxazole Drug Combination ; }, abstract = {Leishmania braziliensis is the main causative agent of Tegumentary Leishmaniasis in the Americas. However, difficulties related to genome manipulation, experimental infection, and parasite growth have so far limited studies with this species. CRISPR-Cas9-based technology has made genome editing more accessible, and here we have successfully employed the LeishGEdit approach to attenuate L. braziliensis. We generated a transgenic cell line expressing Cas9 and T7 RNA polymerase, which was employed for the targeted deletion of centrin, a calcium-binding cytoskeletal protein involved in the centrosome duplication in eukaryotes. Centrin-deficient Leishmania exhibit growth arrest at the amastigote stage. Whole-genome sequencing of centrin-deficient L. braziliensis (LbCen-/-) did not indicate the presence of off-target mutations. In vitro, the growth rates of LbCen-/- and wild-type promastigotes were similar, but axenic and intracellular LbCen-/- amastigotes showed a multinucleated phenotype with impaired survival following macrophage infection. Upon inoculation into BALB/c mice, LbCen-/- were detected at an early time point but failed to induce lesion formation, contrary to control animals, infected with wild-type L. braziliensis. A significantly lower parasite burden was also observed in mice inoculated with LbCen-/- , differently from control mice. Given that centrin-deficient Leishmania sp. have become candidates for vaccine development, we propose that LbCen-/- can be further explored for the purposes of immunoprophylaxis against American Tegumentary Leishmaniasis.}, }
@article {pmid35251616, year = {2022}, author = {Aripova, T and Muratkhodjaev, J}, title = {A novel concept of human antiviral protection: It's all about RNA (Review).}, journal = {Biomedical reports}, volume = {16}, number = {4}, pages = {29}, pmid = {35251616}, issn = {2049-9442}, abstract = {The comparative analysis of the antiviral protective mechanisms, including protozoa and RNA interference in multicellular organisms, has revealed their similarity and provided a basic understanding of adaptive immunity. The present article summarizes the latest studies on RNA-guided gene regulation in human antiviral protection, and its importance. Additionally, the role of both neutralizing antibodies and the interferon system in viral invasion is considered. The interferon system is an additional mechanism for suppressing viral infections in humans, which shifts cells into an 'alarm' mode to attempt to prevent further contagion. The primary task of the human central immune system is to maintain integrity and to protect against foreign organisms. In this review, a novel concept is proposed: Antiviral protection in all organisms can be achieved through an intracellular RNA-guided mechanism. A simple and effective defence against viruses is incorporation of a part of a virus's DNA (spacer) into the hosts chromosomes. Following reinfection, RNA transcripts of this spacer are created to direct nuclease enzymes to destroy the viral genome. This is an example of real-time adaptive immunity potentially possessed by every cell with a full complement of chromosomes, and an indicator that antiviral immunity is not only mediated by the presence of neutralizing antibodies and memory B- and T-cells, but also by the presence of specific spacers in the DNA of individuals who have recovered from a viral infection.}, }
@article {pmid35250902, year = {2021}, author = {Ambroa, A and Blasco, L and López, M and Pacios, O and Bleriot, I and Fernández-García, L and González de Aledo, M and Ortiz-Cartagena, C and Millard, A and Tomás, M}, title = {Genomic Analysis of Molecular Bacterial Mechanisms of Resistance to Phage Infection.}, journal = {Frontiers in microbiology}, volume = {12}, number = {}, pages = {784949}, pmid = {35250902}, issn = {1664-302X}, abstract = {To optimize phage therapy, we need to understand how bacteria evolve against phage attacks. One of the main problems of phage therapy is the appearance of bacterial resistance variants. The use of genomics to track antimicrobial resistance is increasingly developed and used in clinical laboratories. For that reason, it is important to consider, in an emerging future with phage therapy, to detect and avoid phage-resistant strains that can be overcome by the analysis of metadata provided by whole-genome sequencing. Here, we identified genes associated with phage resistance in 18 Acinetobacter baumannii clinical strains belonging to the ST-2 clonal complex during a decade (Ab2000 vs. 2010): 9 from 2000 to 9 from 2010. The presence of genes putatively associated with phage resistance was detected. Genes detected were associated with an abortive infection system, restriction-modification system, genes predicted to be associated with defense systems but with unknown function, and CRISPR-Cas system. Between 118 and 171 genes were found in the 18 clinical strains. On average, 26% of these genes were detected inside genomic islands in the 2000 strains and 32% in the 2010 strains. Furthermore, 38 potential CRISPR arrays in 17 of 18 of the strains were found, as well as 705 proteins associated with CRISPR-Cas systems. A moderately higher presence of these genes in the strains of 2010 in comparison with those of 2000 was found, especially those related to the restriction-modification system and CRISPR-Cas system. The presence of these genes in genomic islands at a higher rate in the strains of 2010 compared with those of 2000 was also detected. Whole-genome sequencing and bioinformatics could be powerful tools to avoid drawbacks when a personalized therapy is applied. In this study, it allows us to take care of the phage resistance in A. baumannii clinical strains to prevent a failure in possible phage therapy.}, }
@article {pmid35250283, year = {2022}, author = {Xu, H and Tang, H and Li, R and Xia, Z and Yang, W and Zhu, Y and Liu, Z and Lu, G and Ni, S and Shen, J}, title = {A New Method Based on LAMP-CRISPR-Cas12a-Lateral Flow Immunochromatographic Strip for Detection.}, journal = {Infection and drug resistance}, volume = {15}, number = {}, pages = {685-696}, pmid = {35250283}, issn = {1178-6973}, abstract = {INTRODUCTION: Carbapenemase-mediated antimicrobial resistance is currently a hot spot of global concern. Carbapenem-resistant organisms are highly prevalent in hospitals associated with difficult-to-treat infections, resulting in poor clinical outcome due to limited treatment options. It is urgently needed to have a rapid, efficient, and convenient molecular assay for identifying such resistant strains.<br><br>METHODS: For this end, we developed a new laboratory assay targeting Klebsiella pneumoniae carbapenemase (KPC) and New Delhi metallo-β-lactamase (NDM) based on loop-mediated isothermal amplification, CRISPR-Cas12a, and lateral flow immunochromatographic strip (CRISPR-Cas-LAMP-lateral flow strip). The method was designed to use a guide RNA (gRNA) to recognize the target DNA and guide Cas12a to cleave the target DNA, and simultaneously cleave any single-stranded DNA within the cleavage reaction system.<br><br>RESULTS: The cleavage products are visible to the naked eye on the lateral flow strip. This method is highly sensitive in direct detection of bacteria in samples containing at least 3×105 CFU/mL without the need for bacterial culture.<br><br>DISCUSSION: It provides shorter turnaround time and higher specificity than the conventional bacterial culture and susceptibility testing method. This new assay is applicable for extensive use in hospital infection control, as well as identification and treatment of resistant strains due to simple operation and inexpensive apparatuses.}, }
@article {pmid35248880, year = {2022}, author = {Kang, B and Cong, Z and Duan, J and Liu, K and Wang, Y}, title = {Generation of a GFI1-flag knock-in human embryonic stem cell line using CRISPR-Cas9 technology.}, journal = {Stem cell research}, volume = {60}, number = {}, pages = {102724}, doi = {10.1016/j.scr.2022.102724}, pmid = {35248880}, issn = {1876-7753}, mesh = {*CRISPR-Cas Systems/genetics ; Cell Line ; DNA-Binding Proteins/genetics/metabolism ; Embryonic Stem Cells/metabolism ; *Human Embryonic Stem Cells/metabolism ; Humans ; Technology ; Transcription Factors/genetics/metabolism ; }, abstract = {GFI1 is a DNA binding transcriptional repressor, it is shown to be an important gene associated with blood cells development and many blood diseases (Möröy et al., 2015). But the role of GFI1 in human hematopoieticdevelopment has not been known (Thambyrajah et al., 2016). To illustrate the function of GFI1 in human hematopoieticdevelopment, we constructed a GFI1-2 × flag-tag knock-in human embryonic stem cell line by CRISPR/Cas9 mediated gene targeting, and it would be the effective tool to study GFI1. The cell line could express GFI1-2 × flag-tag and can be identified with western blot and immunofluorescence. This cell line maintains stem cell morphology, and displays normal karyotype, pluripotent stem cell marker expression and differentiation potential.}, }
@article {pmid35247858, year = {2022}, author = {Safdar, S and Driesen, S and Leirs, K and De Sutter, D and Eyckerman, S and Lammertyn, J and Spasic, D}, title = {Engineered tracrRNA for enabling versatile CRISPR-dCas9-based biosensing concepts.}, journal = {Biosensors &amp; bioelectronics}, volume = {206}, number = {}, pages = {114140}, doi = {10.1016/j.bios.2022.114140}, pmid = {35247858}, issn = {1873-4235}, mesh = {*Biosensing Techniques ; CRISPR-Cas Systems/genetics ; DNA ; *DNA, Catalytic/genetics ; RNA ; }, abstract = {In recent years, CRISPR-Cas (stands for: clustered regularly interspaced short palindromic repeats - CRISPR associated protein) based technologies have gained increasing attention in the biosensing field. Thanks to excellent sequence specificity, their use is of particular interest for detecting nucleic acid (NA) targets. In this context, signal generation and amplification can be realized by employing the cis-cleavage activity of the Cas9 protein, although other options involving the catalytically inactive dead Cas9 (dCas9) are increasingly explored. The latter are however mostly based on complex protein engineering processes and often lack efficient signal amplification. Here we showed for the first time that flexible signal generation and amplification properties can be integrated into the CRISPR-dCas9 complex based on a straightforward incorporation of a DNA sequence into the trans-activating CRISPR RNA (tracrRNA). The intrinsic nuclease activity of the engineered complex remained conserved, while the incorporated DNA stretch enabled two modes of amplified fluorescent signal generation: (1) as an RNA-cleaving DNA-based enzyme (DNAzyme) or (2) as hybridization site for biotinylated DNA probes, allowing subsequent enzyme labeling. Both signal generation strategies were demonstrated in solution as well as while coupled to a solid surface. Finally, in a proof of concept bioassay, we demonstrated the successful detection of single stranded DNA on magnetic microbeads using the engineered CRISPR-dCas9 complex. Thanks to the flexibility of incorporating different NA-based signal generation and amplification strategies, this novel NA engineering approach holds enormous promise for many new CRISPR-based biosensing applications.}, }
@article {pmid35247841, year = {2022}, author = {Chiu, W and Li, A and Wang, T and Li, W and Zhang, X}, title = {Generation of a MSX1 knockout human embryonic stem cell line using CRISPR/Cas9 technology.}, journal = {Stem cell research}, volume = {60}, number = {}, pages = {102729}, doi = {10.1016/j.scr.2022.102729}, pmid = {35247841}, issn = {1876-7753}, mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; *Human Embryonic Stem Cells/metabolism ; Humans ; MSX1 Transcription Factor/genetics/metabolism ; Technology ; }, abstract = {The MSX1 gene encodes a transcriptional repressor and plays important roles in limb-pattern formation, craniofacial development, and odontogenesis during vertebrate embryogenesis. Previous studies demonstrated that human MSX1 mutations are associated with tooth agenesis, orofacial clefting, and nail dysplasia. Here, we generated a MSX1 knockout cell line from human embryonic stem cell (hESC) line (H9) by CRISPR/cas9-mediated gene targeting. This cell line may serve as a valuable in vitro cell model for MSX1 mutation-related diseases and help to gain more insight into the biological function of MSX1.}, }
@article {pmid35247837, year = {2022}, author = {Wang, H and Luo, Y and Li, J and Guan, J and Yang, S and Wang, Q}, title = {Generation of a gene corrected human isogenic iPSC line (CPGHi001-A-1) from a hearing loss patient with the TMC1 p.M418K mutation using CRISPR/Cas9.}, journal = {Stem cell research}, volume = {60}, number = {}, pages = {102736}, doi = {10.1016/j.scr.2022.102736}, pmid = {35247837}, issn = {1876-7753}, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; *Hearing Loss/genetics ; Humans ; *Induced Pluripotent Stem Cells ; Membrane Proteins/genetics ; Mice ; Mutation/genetics ; }, abstract = {TMC1 p.M418K mutation is homologous to that in Beethoven mice, which may induce autosomal dominant non-syndromic progressive hearing loss. Previously, we generated an induced pluripotent stem cells (iPSCs) line (CPGHi001-A) from a hearing loss patient with the TMC1 c.1253 T > A (p.M418K) mutation. Here we genetically corrected the TMC1 c.1253 T > A mutation using CRISPR/Cas9 technology to generate an isogenic control, CPGHi001-A-1. The resulting iPSCs had a normal karyotype, showed pluripotency by immunofluorescence staining, and differentiated into the three germ layers in vitro.}, }
@article {pmid35247836, year = {2022}, author = {Zhang, Y and Zhu, J and Dai, Y and Wang, L and Liu, R and Guo, X}, title = {Generation of a heterozygous FUS-Q290X knock in human embryonic stem cell line (WAe009-A-83) using CRISPR/Cas9 system.}, journal = {Stem cell research}, volume = {60}, number = {}, pages = {102734}, doi = {10.1016/j.scr.2022.102734}, pmid = {35247836}, issn = {1876-7753}, mesh = {*Amyotrophic Lateral Sclerosis/metabolism ; CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism ; Humans ; *Liposarcoma/genetics ; Mutation ; RNA, Messenger/metabolism ; RNA-Binding Protein FUS/genetics/metabolism ; }, abstract = {Fused in Sarcoma (FUS) gene encodes FUS RNA binding protein, a multifunctional protein component of the heterogeneous nuclear ribonucleoprotein complex, which is involved in pre-mRNA splicing and the export of fully processed mRNA to the cytoplasm, and it has been implicated in regulation of gene expression, maintenance of genomic integrity and mRNA/microRNA processing. FUS gene mutations result in amyotrophic lateral sclerosis and Liposarcoma. This heterozygous FUS-Q290X knock in hESC line will be a valuable tool to investigate the disease mechanisms of amyotrophic lateral sclerosis and Liposarcoma.}, }
@article {pmid35247835, year = {2022}, author = {Bray, L and Caillaud, A and Girardeau, A and Patitucci, M and Le May, C and Cariou, B and Rimbert, A}, title = {Generation of a GPR146 knockout human induced pluripotent stem cell line (ITXi001-A-1).}, journal = {Stem cell research}, volume = {60}, number = {}, pages = {102721}, doi = {10.1016/j.scr.2022.102721}, pmid = {35247835}, issn = {1876-7753}, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Liver ; Mice ; }, abstract = {Dyslipidemia is a key modifiable causal risk factor involved in the development of atherosclerotic cardiovascular disease. Recently, the G protein-coupled receptor 146 (GPR146), a member of the G-coupled protein receptors' family, has been shown to be a regulator of plasma cholesterol. Inhibition of hepatic GPR146 in mice displays protective effect against both hypercholesterolemia and atherosclerosis. Here, we characterize a genetically engineered human induced pluripotent stem cell (hiPSC) model invalidated for GPR146 (ITXi001-A-1) using CRISPR-Cas9 editing technology. Differentiation of ITXi001-A-1 towards hepatic fate will provide a suitable model for deciphering the molecular mechanisms sustaining the beneficial metabolic effects of GPR146 inhibition.}, }
@article {pmid35246186, year = {2022}, author = {Guo, M and Chen, H and Dong, S and Zhang, Z and Luo, H}, title = {CRISPR-Cas gene editing technology and its application prospect in medicinal plants.}, journal = {Chinese medicine}, volume = {17}, number = {1}, pages = {33}, pmid = {35246186}, issn = {1749-8546}, abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas gene editing technology has opened a new era of genome interrogation and genome engineering because of its ease operation and high efficiency. An increasing number of plant species have been subjected to site-directed gene editing through this technology. However, the application of CRISPR-Cas technology to medicinal plants is still in the early stages. Here, we review the research history, structural characteristics, working mechanism and the latest derivatives of CRISPR-Cas technology, and discussed their application in medicinal plants for the first time. Furthermore, we creatively put forward the development direction of CRISPR technology applied to medicinal plant gene editing. The aim is to provide a reference for the application of this technology to genome functional studies, synthetic biology, genetic improvement, and germplasm innovation of medicinal plants. CRISPR-Cas is expected to revolutionize medicinal plant biotechnology in the near future.}, }
@article {pmid35245852, year = {2022}, author = {Zhang, F and Meier, AB and Lipp, P and Laugwitz, KL and Dorn, T and Moretti, A}, title = {Generation of heterozygous (MRli003-A-3) and homozygous (MRli003-A-4) TRPM4 knockout human iPSC lines.}, journal = {Stem cell research}, volume = {60}, number = {}, pages = {102731}, doi = {10.1016/j.scr.2022.102731}, pmid = {35245852}, issn = {1876-7753}, support = {788381/ERC_/European Research Council/International ; }, mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; Heterozygote ; Homozygote ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Mutation/genetics ; *TRPM Cation Channels/genetics/metabolism ; }, abstract = {TRPM4 is a Ca2+-activated channel mediating the transport of monovalent cations across the cell membrane. Mutations in the TRPM4 gene have been associated with cardiac arrhythmias in humans. Using CRISPR/Cas9 gene editing technology, we established two TRPM4 knockout human iPSC lines - one heterozygous (MRli003-A-3) and one homozygous (MRli003-A-4) - by inserting a frameshift mutation in exon 2 of the TRPM4 gene. Both lines maintained pluripotency, a normal karyotype, parental cell morphology, and the ability to differentiate into the three germ layers.}, }
@article {pmid35245313, year = {2022}, author = {Wen, T and Mao, C and Gao, L}, title = {Analysis of the gut microbiota composition of myostatin mutant cattle prepared using CRISPR/Cas9.}, journal = {PloS one}, volume = {17}, number = {3}, pages = {e0264849}, pmid = {35245313}, issn = {1932-6203}, mesh = {Animals ; Bacteria/genetics ; CRISPR-Cas Systems ; Cattle ; *Gastrointestinal Microbiome/genetics ; Myostatin/genetics ; RNA, Ribosomal, 16S/genetics ; }, abstract = {Myostatin (MSTN) negatively regulates muscle development and positively regulates metabolism through various pathways. Although MSTN function in cattle has been widely studied, the changes in the gut microbiota due to MSTN mutation, which contribute to host health by regulating its metabolism, remain unclear. Here, high-throughput sequencing of the 16S rRNA gene was conducted to analyze the gut microbiota of wild-type (WT) and MSTN mutant (MT) cattle. A total of 925 operational taxonomic units (OTUs) were obtained, which were classified into 11 phyla and 168 genera. Alpha diversity results showed no significant differences between MT and WT cattle. Beta diversity analyses suggested that the microbial composition of WT and MT cattle was different. Three dominant phyla and 21 dominant genera were identified. The most abundant bacterial genus had a significant relationship with the host metabolism. Moreover, various bacteria beneficial for health were found in the intestines of MT cattle. Analysis of the correlation between dominant gut bacteria and serum metabolic factors affected by MSTN mutation indicated that MSTN mutation affected the metabolism mainly by three metabolism-related bacteria, Ruminococcaceae_UCG-013, Clostridium_sensu_stricto_1, and Ruminococcaceae_UCG-010. This study provides further insight into MSTN mutation regulating the host metabolism by gut microbes and provides evidence for the safety of gene-edited animals.}, }
@article {pmid35245307, year = {2022}, author = {Zhang, H and Zhu, S and Xing, Y and Liu, Q and Guo, Z and Cai, Z and Shen, Z and Xia, Q and Sheng, H}, title = {Effect of cryopreservation on A172 and U251 glioma cells infected with lentiviral vectors designed for CRISPR/Cas9-mediated aquaporin-8 knock-out.}, journal = {PloS one}, volume = {17}, number = {3}, pages = {e0263162}, pmid = {35245307}, issn = {1932-6203}, mesh = {*Aquaporins/genetics ; CRISPR-Cas Systems/genetics ; Cryopreservation ; Gene Editing/methods ; *Glioma ; Humans ; Lentivirus/genetics ; RNA, Guide/genetics ; RNA, Messenger ; Reproducibility of Results ; }, abstract = {Among the three existing targeted gene editing technologies, zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats-CRISPR-associated 9 (CRISPR-Cas9), the latter is widely used owing to its simplicity, efficiency, and low cost. Here, we routinely infected A172 and U251 cells with lentiviral vectors, in which aquaporin-8 (AQP8) was knocked out using CRISPR/Cas9. Our results indicated that cryopreservation did not significantly alter the viral infection efficiency, but influenced AQP8 expression in the infected cells at both protein and mRNA levels compared with the non-cryopreserved samples. Further, AQP8 expression at protein and mRNA levels in recovered cryopreserved infected cells did not significantly differ from those in the blank and negative controls, indicating that the lentivirus was still infectious at low temperatures. However, it failed to release the AQP8-targeting guide RNA in the infected cells, or the guide RNA was released, but underwent changes that caused it to malfunction in the cells with CRISPR/Cas9-mediated AQP8 knock-out. Our findings possibly provide some insights into the reliability of lentiviruses as CRISPR/Cas9 vectors.}, }
@article {pmid35244719, year = {2022}, author = {Liu, Y and Ma, G and Gao, Z and Li, J and Wang, J and Zhu, X and Ma, R and Yang, J and Zhou, Y and Hu, K and Zhang, Y and Guo, Y}, title = {Global chromosome rearrangement induced by CRISPR-Cas9 reshapes the genome and transcriptome of human cells.}, journal = {Nucleic acids research}, volume = {50}, number = {6}, pages = {3456-3474}, pmid = {35244719}, issn = {1362-4962}, mesh = {CRISPR-Cas Systems ; Chromosomes/metabolism ; DNA Copy Number Variations ; *Gene Editing/methods ; Genome, Human ; Humans ; RNA, Guide/genetics ; *Transcriptome ; }, abstract = {Chromosome rearrangement plays important roles in development, carcinogenesis and evolution. However, its mechanism and subsequent effects are not fully understood. Large-scale chromosome rearrangement has been performed in the simple eukaryote, wine yeast, but the relative research in mammalian cells remains at the level of individual chromosome rearrangement due to technical limitations. In this study, we used CRISPR-Cas9 to target the highly repetitive human endogenous retrotransposons, LINE-1 and Alu, resulting in a large number of DNA double-strand breaks in the chromosomes. While this operation killed the majority of the cells, we eventually obtained live cell groups. Karyotype analysis and genome re-sequencing proved that we have achieved global chromosome rearrangement (GCR) in human cells. The copy number variations of the GCR genomes showed typical patterns observed in tumor genomes. The ATAC-seq and RNA-seq further revealed that the epigenetic and transcriptomic landscapes were deeply reshaped by GCR. Gene expressions related to p53 pathway, DNA repair, cell cycle and apoptosis were greatly altered to facilitate the cell survival. Our study provided a new application of CRISPR-Cas9 and a practical approach for GCR in complex mammalian genomes.}, }
@article {pmid35244489, year = {2022}, author = {Nguyen Tran, MT and Kc, R and Hewitt, AW}, title = {A Taxonomic and Phylogenetic Classification of Diverse Base Editors.}, journal = {The CRISPR journal}, volume = {5}, number = {2}, pages = {311-328}, doi = {10.1089/crispr.2021.0095}, pmid = {35244489}, issn = {2573-1602}, mesh = {*CRISPR-Cas Systems ; DNA/genetics ; *Gene Editing ; Phylogeny ; }, abstract = {Base editors mediate the targeted conversion of single nucleobases in a therapeutically relevant manner. Herein, we present a hypothetical taxonomic and phylogenetic framework for the classification of more than 200 different DNA base editors, and we categorize them based on their described properties. Following evaluation of their in situ activity windows, which were derived by cataloguing their activity in published literature, organization is done hierarchically, with specific base editor signatures being subcategorized according to their on-target activity or nonspecific, genome- or transcriptome-wide activity. Based on this categorization, we curate a phylogenetic framework, based on protein homology alignment, and describe a taxonomic structure that clusters base editor variants on their target chemistry, endonuclease component, identity of their deaminase component, and their described properties into discrete taxa. Thus, we establish a hypothetical taxonomic structure that can describe and organize current and potentially future base editing variants into clearly defined groups that are defined by their characteristics. Finally, we summarize our findings into a navigable database (ShinyApp in R) that allows users to select through our repository to nominate ideal base editor candidates as a starting point for further testing in their specific application.}, }
@article {pmid35244184, year = {2022}, author = {Thiruppathi, D}, title = {CRISPR keeps going "wild": a new protocol for DNA-free genome editing of tetraploid wild tomatoes.}, journal = {Plant physiology}, volume = {189}, number = {1}, pages = {10-11}, pmid = {35244184}, issn = {1532-2548}, mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/methods ; *Lycopersicon esculentum/genetics ; *Solanum/genetics ; Tetraploidy ; }, }
@article {pmid35243691, year = {2022}, author = {Thompson, EL and Pickett-Leonard, M and Riddle, MJ and Chen, W and Albert, FW and Tolar, J}, title = {Genes and compounds that increase type VII collagen expression as potential treatments for dystrophic epidermolysis bullosa.}, journal = {Experimental dermatology}, volume = {}, number = {}, pages = {}, doi = {10.1111/exd.14555}, pmid = {35243691}, issn = {1600-0625}, support = {/AR/NIAMS NIH HHS/United States ; /NH/NIH HHS/United States ; //Children's Cancer Research Fund/ ; /AG/NIA NIH HHS/United States ; T32-AG029796//Dr. Thompson is partly funded by NIA Training/ ; R21-HG010380//Drs. Tolar and Albert are partly supported by NIH/ ; R01-AR063070//Dr. Tolar is partly supported by NIH/NIAMS/ ; }, abstract = {Dystrophic epidermolysis bullosa (DEB) is a skin-blistering disease caused by mutations in COL7A1, which encodes type VII collagen (C7). There is no cure for DEB, but previous work has shown potential therapeutic benefit of increased production of even partially functional C7. Genome-wide screens using CRISPR-Cas9 have enabled the identification of genes involved in cancer development, drug resistance and other genetic diseases, suggesting that they could be used to identify drivers of C7 production. A keratinocyte C7 reporter cell line was created and used in a genome-wide CRISPR activation (CRISPRa) screen to identify genes and pathways that increase C7 expression. The CRISPRa screen results were used to develop a targeted drug screen to identify compounds that upregulate C7 expression. The C7_tdTomato cell line was validated as an effective reporter for detection of C7 upregulation. The CRISPRa screen identified DENND4B and TYROBP as top gene hits plus pathways related to calcium uptake and immune signalling in C7 regulation. The targeted drug screen identified several compounds that increase C7 expression in keratinocytes, of which kaempferol, a plant flavonoid, also significantly increased C7 mRNA and protein in DEB patient cells.}, }
@article {pmid35241090, year = {2022}, author = {Rasul, MF and Hussen, BM and Salihi, A and Ismael, BS and Jalal, PJ and Zanichelli, A and Jamali, E and Baniahmad, A and Ghafouri-Fard, S and Basiri, A and Taheri, M}, title = {Strategies to overcome the main challenges of the use of CRISPR/Cas9 as a replacement for cancer therapy.}, journal = {Molecular cancer}, volume = {21}, number = {1}, pages = {64}, pmid = {35241090}, issn = {1476-4598}, mesh = {*CRISPR-Cas Systems ; Gene Editing ; Genetic Therapy ; Humans ; Mutation ; *Neoplasms/genetics/therapy ; }, abstract = {CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-associated protein 9) shows the opportunity to treat a diverse array of untreated various genetic and complicated disorders. Therapeutic genome editing processes that target disease-causing genes or mutant genes have been greatly accelerated in recent years as a consequence of improvements in sequence-specific nuclease technology. However, the therapeutic promise of genome editing has yet to be explored entirely, many challenges persist that increase the risk of further mutations. Here, we highlighted the main challenges facing CRISPR/Cas9-based treatments and proposed strategies to overcome these limitations, for further enhancing this revolutionary novel therapeutics to improve long-term treatment outcome human health.}, }
@article {pmid35239244, year = {2022}, author = {Shimizu, S and Shiraki, A}, title = {CRISPR/Cas9 unveils the dynamics of the endogenous µ-opioid receptors on neuronal cells under continuous opioid stimulation.}, journal = {Pharmacology research &amp; perspectives}, volume = {10}, number = {2}, pages = {e00933}, pmid = {35239244}, issn = {2052-1707}, mesh = {*Analgesics, Opioid/pharmacology ; CRISPR-Cas Systems ; Ligands ; Receptors, Opioid ; *Receptors, Opioid, mu/genetics/metabolism ; }, abstract = {Long-term opioid use develops tolerance and attenuates analgesic effects. Upon activation, µ-opioid receptors (MOPs) are internalized and directed to either recycling or degradation pathway. Ligand stimulation also promotes de novo MOP synthesis. These processes collaboratively regulate MOP expression and play critical roles in tolerance development. However, there is limited understanding of how the endogenous MOP expression changes after prolonged opioid administration because previous analyses have focused on individual processes using overexpression systems, which ignored physiological regulation. Another fundamental problem is the unavailability of commercial antibodies to detect the low expression of endogenous MOP in neuronal systems. Here, we established a neuronal cell line to detect endogenous MOP with sufficient sensitivity using CRISPR/Cas9 technology. We incorporated the hemagglutinin sequence into the MOP gene of the SH-SY5Y cell. The genome-editing did not significantly impair MOP functions such as MOP internalization or the downstream signaling. The clone was differentiated into a state similar to the primary culture undergoing treatment with all-trans retinoic acid, followed by brain-derived neurotrophic factor. Upon continuous stimulation with MOP ligands, endogenous MOP constantly decreased up to 48 h. The expression level was maintained at a certain level following this period, depending on the ligand properties. DAMGO reduced MOP from the cell surface by about 70%, while morphine did so by 40%. Our results indicate that even a few days of opioid administration could significantly reduce the MOP expression level. Our cell line could be a potential tool to investigate the molecular mechanisms underlying the problems caused by long-term opioid use.}, }
@article {pmid35238621, year = {2022}, author = {Yu, SY and Birkenshaw, A and Thomson, T and Carlaw, T and Zhang, LH and Ross, CJD}, title = {Increasing the Targeting Scope of CRISPR Base Editing System Beyond NGG.}, journal = {The CRISPR journal}, volume = {5}, number = {2}, pages = {187-202}, doi = {10.1089/crispr.2021.0109}, pmid = {35238621}, issn = {2573-1602}, support = {NMIN 2019-T2-05//CIHR/Canada ; }, mesh = {*CRISPR-Cas Systems/genetics ; DNA ; DNA Breaks, Double-Stranded ; *Gene Editing ; Humans ; Nucleotides ; }, abstract = {Genome editing provides a new therapeutic strategy to cure genetic diseases. The recently developed CRISPR-Cas9 base editing technology has shown great potential to repair the majority of pathogenic point mutations in the patient's DNA precisely. Base editor is the fusion of a Cas9 nickase with a base-modifying enzyme that can change a nucleotide on a single strand of DNA without generating double-stranded DNA breaks. However, a major limitation in applying such a system is the prerequisite of a protospacer adjacent motif sequence at the desired position relative to the target site. Progress has been made to increase the targeting scope of base editors by engineering SpCas9 protein variants, establishing systems with broadened editing windows, characterizing new SpCas9 orthologs, and developing prime editing technology. In this review, we discuss recent progress in the development of CRISPR base editing, focusing on its targeting scope, and we provide a workflow for selecting a suitable base editor based on the target nucleotide sequences.}, }
@article {pmid35238530, year = {2022}, author = {Nouri, R and Dong, M and Politza, AJ and Guan, W}, title = {Figure of Merit for CRISPR-Based Nucleic Acid-Sensing Systems: Improvement Strategies and Performance Comparison.}, journal = {ACS sensors}, volume = {7}, number = {3}, pages = {900-911}, doi = {10.1021/acssensors.2c00024}, pmid = {35238530}, issn = {2379-3694}, support = {R61 AI147419/AI/NIAID NIH HHS/United States ; }, mesh = {Biological Assay ; CRISPR-Cas Systems/genetics ; *Nucleic Acids ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid-sensing systems have grown rapidly in the past few years. Nevertheless, an objective approach to benchmark the performances of different CRISPR sensing systems is lacking due to the heterogeneous experimental setup. Here, we developed a quantitative CRISPR sensing figure of merit (FOM) to compare different CRISPR methods and explore performance improvement strategies. The CRISPR sensing FOM is defined as the product of the limit of detection (LOD) and the associated CRISPR reaction time (T). A smaller FOM means that the method can detect smaller target quantities faster. We found that there is a tradeoff between the LOD of the assay and the required reaction time. With the proposed CRISPR sensing FOM, we evaluated five strategies to improve the CRISPR-based sensing: preamplification, enzymes of higher catalytic efficiency, multiple crRNAs, digitalization, and sensitive readout systems. We benchmarked the FOM performances of 57 existing studies and found that the effectiveness of these strategies on improving the FOM is consistent with the model prediction. In particular, we found that digitalization is the most promising amplification-free method for achieving comparable FOM performances (∼1 fM·min) as those using preamplification. The findings here would have broad implications for further optimization of the CRISPR-based sensing.}, }
@article {pmid35238390, year = {2022}, author = {Chen, J and Li, S and He, Y and Li, J and Xia, L}, title = {An update on precision genome editing by homology-directed repair in plants.}, journal = {Plant physiology}, volume = {188}, number = {4}, pages = {1780-1794}, pmid = {35238390}, issn = {1532-2548}, mesh = {*CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; DNA Repair/genetics ; *Gene Editing ; Recombinational DNA Repair/genetics ; }, abstract = {Beneficial alleles derived from local landraces or related species, or even orthologs from other plant species, are often caused by differences of one or several single-nucleotide polymorphisms or indels in either the promoter region or the encoding region of a gene and often account for major differences in agriculturally important traits. Clustered regularly interspaced short palindromic repeats-associated endonuclease Cas9 system (CRISPR/Cas9)-mediated precision genome editing enables targeted allele replacement or insertion of flag or foreign genes at specific loci via homology-directed repair (HDR); however, HDR efficiency is low due to the intrinsic rare occurrence of HDR and insufficient DNA repair template in the proximity of a double-stranded break (DSB). Precise replacement of the targeted gene with elite alleles from landraces or relatives into a commercial variety through genome editing has been a holy grail in the crop genome editing field. In this update, we briefly summarize CRISPR/Cas-mediated HDR in plants. We describe diverse strategies to improve HDR efficiency by manipulating the DNA repair pathway, timing DSB induction, and donor delivery, and so on. Lastly, we outline open questions and challenges in HDR-mediated precision genome editing in both plant biological research and crop improvement.}, }
@article {pmid35237996, year = {2022}, author = {De Giorgi, M and Jarrett, KE and de Aguiar Vallim, TQ and Lagor, WR}, title = {In Vivo Gene Editing in Lipid and Atherosclerosis Research.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2419}, number = {}, pages = {673-713}, pmid = {35237996}, issn = {1940-6029}, support = {U42 OD026645/OD/NIH HHS/United States ; UG3 HL151545/HL/NHLBI NIH HHS/United States ; T32 HL069766/HL/NHLBI NIH HHS/United States ; }, mesh = {Animals ; *Atherosclerosis/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Hypercholesterolemia/genetics ; *Hyperlipidemias ; Mice ; }, abstract = {The low-density lipoprotein receptor (Ldlr) and apolipoprotein E (Apoe) germline knockout (KO) models have provided fundamental insights in lipid and atherosclerosis research for decades. However, testing new candidate genes in these models requires extensive breeding, which is highly time and resource consuming. In this chapter, we provide methods for rapidly modeling hypercholesterolemia and atherosclerosis as well as testing new genes in adult mice through somatic gene editing. Adeno-associated viral (AAV) vectors are exploited to deliver the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing system (AAV-CRISPR) to the liver. This tool enables rapid and efficient editing of lipid- and atherosclerosis-related genes in the liver.}, }
@article {pmid35237980, year = {2022}, author = {Xian, X and Wang, Y and Liu, G}, title = {Genetically Engineered Hamster Models of Dyslipidemia and Atherosclerosis.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2419}, number = {}, pages = {433-459}, pmid = {35237980}, issn = {1940-6029}, mesh = {Animals ; Animals, Genetically Modified ; *Atherosclerosis/genetics ; CRISPR-Cas Systems ; Cricetinae ; Disease Models, Animal ; *Dyslipidemias/genetics ; Genetic Engineering/methods ; Mice ; Rabbits ; Rats ; Swine ; }, abstract = {Animal models of human diseases play an extremely important role in biomedical research. Among them, mice are widely used animal models for translational research, especially because of ease of generation of genetically engineered mice. However, because of the great differences in biology between mice and humans, translation of findings to humans remains a major issue. Therefore, the exploration of models with biological and metabolic characteristics closer to those of humans has never stopped.Although pig and nonhuman primates are biologically similar to humans, their genetic engineering is technically difficult, the cost of breeding is high, and the experimental time is long. As a result, the application of these species as model animals, especially genetically engineered model animals, in biomedical research is greatly limited.In terms of lipid metabolism and cardiovascular diseases, hamsters have several characteristics different from rats and mice, but similar to those in humans. The hamster is therefore an ideal animal model for studying lipid metabolism and cardiovascular disease because of its small size and short reproduction period. However, the phenomenon of zygote division, which was unexpectedly blocked during the manipulation of hamster embryos for some unknown reasons, had plagued researchers for decades and no genetically engineered hamsters have therefore been generated as animal models of human diseases for a long time. After solving the problem of in vitro development of hamster zygotes, we successfully prepared enhanced green fluorescent protein (eGFP) transgenic hamsters by microinjection of lentiviral vectors into the zona pellucida space of zygotes. On this basis, we started the development of cardiovascular disease models using the hamster embryo culture system combined with the novel genome editing technique of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9). In this chapter, we will introduce some of the genetically engineered hamster models with dyslipidemia and the corresponding characteristics of these models. We hope that the genetically engineered hamster models can be further recognized and complement other genetically engineered animal models such as mice, rats, and rabbits. This will lead to new avenues and pathways for the study of lipid metabolism and its related diseases.}, }
@article {pmid35237818, year = {2021}, author = {Guiziou, S and Chu, JC and Nemhauser, JL}, title = {Decoding and recoding plant development.}, journal = {Plant physiology}, volume = {187}, number = {2}, pages = {515-526}, pmid = {35237818}, issn = {1532-2548}, support = {/HHMI/Howard Hughes Medical Institute/United States ; }, mesh = {CRISPR-Cas Systems/genetics ; *Cell Differentiation ; *Cell Lineage ; *Gene Editing ; Genetic Engineering ; Integrases/genetics ; Molecular Biology ; Plant Development/genetics/*physiology ; *Synthetic Biology ; *Systems Biology ; }, abstract = {The development of multicellular organisms has been studied for centuries, yet many critical events and mechanisms of regulation remain challenging to observe directly. Early research focused on detailed observational and comparative studies. Molecular biology has generated insights into regulatory mechanisms, but only for a limited number of species. Now, synthetic biology is bringing these two approaches together, and by adding the possibility of sculpting novel morphologies, opening another path to understanding biology. Here, we review a variety of recently invented techniques that use CRISPR/Cas9 and phage integrases to trace the differentiation of cells over various timescales, as well as to decode the molecular states of cells in high spatiotemporal resolution. Most of these tools have been implemented in animals. The time is ripe for plant biologists to adopt and expand these approaches. Here, we describe how these tools could be used to monitor development in diverse plant species, as well as how they could guide efforts to recode programs of interest.}, }
@article {pmid35237805, year = {2021}, author = {Ali, Z and Mahfouz, MM}, title = {CRISPR/Cas systems versus plant viruses: engineering plant immunity and beyond.}, journal = {Plant physiology}, volume = {186}, number = {4}, pages = {1770-1785}, pmid = {35237805}, issn = {1532-2548}, mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/genetics/virology ; Disease Resistance/*genetics ; Gene Editing/methods ; Plant Breeding/*methods ; Plant Diseases/*genetics/*virology ; Plant Immunity/*genetics ; Plant Viruses/*pathogenicity ; }, abstract = {Molecular engineering of plant immunity to confer resistance against plant viruses holds great promise for mitigating crop losses and improving plant productivity and yields, thereby enhancing food security. Several approaches have been employed to boost immunity in plants by interfering with the transmission or lifecycles of viruses. In this review, we discuss the successful application of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) (CRISPR/Cas) systems to engineer plant immunity, increase plant resistance to viruses, and develop viral diagnostic tools. Furthermore, we examine the use of plant viruses as delivery systems to engineer virus resistance in plants and provide insight into the limitations of current CRISPR/Cas approaches and the potential of newly discovered CRISPR/Cas systems to engineer better immunity and develop better diagnostics tools for plant viruses. Finally, we outline potential solutions to key challenges in the field to enable the practical use of these systems for crop protection and viral diagnostics.}, }
@article {pmid35236982, year = {2022}, author = {Bravo, JPK and Liu, MS and Hibshman, GN and Dangerfield, TL and Jung, K and McCool, RS and Johnson, KA and Taylor, DW}, title = {Structural basis for mismatch surveillance by CRISPR-Cas9.}, journal = {Nature}, volume = {603}, number = {7900}, pages = {343-347}, pmid = {35236982}, issn = {1476-4687}, mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Cryoelectron Microscopy ; DNA/chemistry/genetics ; *DNA Mismatch Repair ; *Gene Editing ; Nucleic Acid Conformation ; *RNA, Guide/genetics ; }, abstract = {CRISPR-Cas9 as a programmable genome editing tool is hindered by off-target DNA cleavage1-4, and the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood5-7. Although Cas9 variants with greater discrimination against mismatches have been designed8-10, these suffer from substantially reduced rates of on-target DNA cleavage5,11. Here we used kinetics-guided cryo-electron microscopy to determine the structure of Cas9 at different stages of mismatch cleavage. We observed a distinct, linear conformation of the guide RNA-DNA duplex formed in the presence of mismatches, which prevents Cas9 activation. Although the canonical kinked guide RNA-DNA duplex conformation facilitates DNA cleavage, we observe that substrates that contain mismatches distal to the protospacer adjacent motif are stabilized by reorganization of a loop in the RuvC domain. Mutagenesis of mismatch-stabilizing residues reduces off-target DNA cleavage but maintains rapid on-target DNA cleavage. By targeting regions that are exclusively involved in mismatch tolerance, we provide a proof of concept for the design of next-generation high-fidelity Cas9 variants.}, }
@article {pmid35236841, year = {2022}, author = {Smargon, AA and Madrigal, AA and Yee, BA and Dong, KD and Mueller, JR and Yeo, GW}, title = {Crosstalk between CRISPR-Cas9 and the human transcriptome.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {1125}, pmid = {35236841}, issn = {2041-1723}, support = {R01 EY029166/EY/NEI NIH HHS/United States ; R01 HG004659/HG/NHGRI NIH HHS/United States ; R01 NS103172/NS/NINDS NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; RNA Editing ; *RNA, Guide/metabolism ; Transcriptome ; }, abstract = {CRISPR-Cas9 expression independent of its cognate synthetic guide RNA (gRNA) causes widespread genomic DNA damage in human cells. To investigate whether Cas9 can interact with endogenous human RNA transcripts independent of its guide, we perform eCLIP (enhanced CLIP) of Cas9 in human cells and find that Cas9 reproducibly interacts with hundreds of endogenous human RNA transcripts. This association can be partially explained by a model built on gRNA secondary structure and sequence. Critically, transcriptome-wide Cas9 binding sites do not appear to correlate with published genome-wide Cas9 DNA binding or cut-site loci under gRNA co-expression. However, even under gRNA co-expression low-affinity Cas9-human RNA interactions (which we term CRISPR crosstalk) do correlate with published elevated transcriptome-wide RNA editing. Our findings do not support the hypothesis that human RNAs can broadly guide Cas9 to bind and cleave human genomic DNA, but they illustrate a cellular and RNA impact likely inherent to CRISPR-Cas systems.}, }
@article {pmid35236570, year = {2022}, author = {Lee, H and Sashital, DG}, title = {Creating memories: molecular mechanisms of CRISPR adaptation.}, journal = {Trends in biochemical sciences}, volume = {47}, number = {6}, pages = {464-476}, doi = {10.1016/j.tibs.2022.02.004}, pmid = {35236570}, issn = {0968-0004}, support = {R01 GM115874/GM/NIGMS NIH HHS/United States ; R35 GM140876/GM/NIGMS NIH HHS/United States ; }, mesh = {*CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems ; }, abstract = {Prokaryotes use clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas) proteins as an adaptive immune system. CRISPR-Cas systems preserve molecular memories of infections by integrating short fragments of foreign nucleic acids as spacers into the host CRISPR array in a process termed 'adaptation'. Functional spacers ensure a robust immune response by Cas effectors, which neutralizes subsequent infection through RNA-guided interference pathways. In this review, we summarize recent discoveries that have advanced our understanding of adaptation, with a focus on how functional spacers are generated and incorporated through many widespread, but type-specific, mechanisms. Finally, we highlight future directions and outstanding questions for a more thorough understanding of CRISPR adaptation.}, }
@article {pmid35236300, year = {2022}, author = {Krohannon, A and Srivastava, M and Rauch, S and Srivastava, R and Dickinson, BC and Janga, SC}, title = {CASowary: CRISPR-Cas13 guide RNA predictor for transcript depletion.}, journal = {BMC genomics}, volume = {23}, number = {1}, pages = {172}, pmid = {35236300}, issn = {1471-2164}, support = {R01 GM123314/GM/NIGMS NIH HHS/United States ; R01 MH122142/MH/NIMH NIH HHS/United States ; R35 GM119840/GM/NIGMS NIH HHS/United States ; R01GM123314/GM/NIGMS NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems ; Gene Editing/methods ; HEK293 Cells ; HeLa Cells ; Humans ; *RNA, Guide/genetics ; Reproducibility of Results ; }, abstract = {BACKGROUND: Recent discovery of the gene editing system - CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) associated proteins (Cas), has resulted in its widespread use for improved understanding of a variety of biological systems. Cas13, a lesser studied Cas protein, has been repurposed to allow for efficient and precise editing of RNA molecules. The Cas13 system utilizes base complementarity between a crRNA/sgRNA (crispr RNA or single guide RNA) and a target RNA transcript, to preferentially bind to only the target transcript. Unlike targeting the upstream regulatory regions of protein coding genes on the genome, the transcriptome is significantly more redundant, leading to many transcripts having wide stretches of identical nucleotide sequences. Transcripts also exhibit complex three-dimensional structures and interact with an array of RBPs (RNA Binding Proteins), both of which may impact the effectiveness of transcript depletion of target sequences. However, our understanding of the features and corresponding methods which can predict whether a specific sgRNA will effectively knockdown a transcript is very limited.<br><br>RESULTS: Here we present a novel machine learning and computational tool, CASowary, to predict the efficacy of a sgRNA. We used publicly available RNA knockdown data from Cas13 characterization experiments for 555 sgRNAs targeting the transcriptome in HEK293 cells, in conjunction with transcriptome-wide protein occupancy information. Our model utilizes a Decision Tree architecture with a set of 112 sequence and target availability features, to classify sgRNA efficacy into one of four classes, based upon expected level of target transcript knockdown. After accounting for noise in the training data set, the noise-normalized accuracy exceeds 70%. Additionally, highly effective sgRNA predictions have been experimentally validated using an independent RNA targeting Cas system - CIRTS, confirming the robustness and reproducibility of our model's sgRNA predictions. Utilizing transcriptome wide protein occupancy map generated using POP-seq in HeLa cells against publicly available protein-RNA interaction map in Hek293 cells, we show that CASowary can predict high quality guides for numerous transcripts in a cell line specific manner.<br><br>CONCLUSIONS: Application of CASowary to whole transcriptomes should enable rapid deployment of CRISPR/Cas13 systems, facilitating the development of therapeutic interventions linked with aberrations in RNA regulatory processes.}, }
@article {pmid35235462, year = {2022}, author = {Bakkers, MJG and Moon-Walker, A and Herlo, R and Brusic, V and Stubbs, SH and Hastie, KM and Saphire, EO and Kirchhausen, TL and Whelan, SPJ}, title = {CD164 is a host factor for lymphocytic choriomeningitis virus entry.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {119}, number = {10}, pages = {e2119676119}, pmid = {35235462}, issn = {1091-6490}, support = {F31 AI154700/AI/NIAID NIH HHS/United States ; T32 AI007245/AI/NIAID NIH HHS/United States ; T32 AI125179/AI/NIAID NIH HHS/United States ; U19 AI109740/AI/NIAID NIH HHS/United States ; }, mesh = {A549 Cells ; CRISPR-Cas Systems ; Endolyn/physiology ; Gene Editing ; HEK293 Cells ; HeLa Cells ; Host-Pathogen Interactions ; Humans ; Hydrogen-Ion Concentration ; Lymphocytic choriomeningitis virus/pathogenicity/*physiology ; Membrane Fusion ; Virulence Factors ; *Virus Internalization ; }, abstract = {Significance Lymphocytic choriomeningitis virus (LCMV) is the prototypic arenavirus and has been utilized for decades as a model to understand the host immune response against viral infection. LCMV infection can lead to fatal meningitis in immunocompromised people and can lead to congenital birth defects and spontaneous abortion if acquired during pregnancy. Using a genetic screen, we uncover host factors involved in LCMV entry that were previously unknown and are candidate therapeutic targets to combat LCMV infection. This study expands our understanding of the entry pathway of LCMV, revealing that its glycoprotein switches from utilizing the known receptor α-DG and heparan sulfate at the plasma membrane to binding the lysosomal mucin CD164 at pH levels found in endolysosomal compartments, facilitating membrane fusion.}, }
@article {pmid35235150, year = {2022}, author = {Li, G and Li, X and Zhuang, S and Wang, L and Zhu, Y and Chen, Y and Sun, W and Wu, Z and Zhou, Z and Chen, J and Huang, X and Wang, J and Li, D and Li, W and Wang, H and Wei, W}, title = {Gene editing and its applications in biomedicine.}, journal = {Science China. Life sciences}, volume = {65}, number = {4}, pages = {660-700}, pmid = {35235150}, issn = {1869-1889}, mesh = {*CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Endonucleases/genetics ; *Gene Editing/methods ; Humans ; }, abstract = {The steady progress in genome editing, especially genome editing based on the use of clustered regularly interspaced short palindromic repeats (CRISPR) and programmable nucleases to make precise modifications to genetic material, has provided enormous opportunities to advance biomedical research and promote human health. The application of these technologies in basic biomedical research has yielded significant advances in identifying and studying key molecular targets relevant to human diseases and their treatment. The clinical translation of genome editing techniques offers unprecedented biomedical engineering capabilities in the diagnosis, prevention, and treatment of disease or disability. Here, we provide a general summary of emerging biomedical applications of genome editing, including open challenges. We also summarize the tools of genome editing and the insights derived from their applications, hoping to accelerate new discoveries and therapies in biomedicine.}, }
@article {pmid35234927, year = {2022}, author = {O'Geen, H and Tomkova, M and Combs, JA and Tilley, EK and Segal, DJ}, title = {Determinants of heritable gene silencing for KRAB-dCas9 + DNMT3 and Ezh2-dCas9 + DNMT3 hit-and-run epigenome editing.}, journal = {Nucleic acids research}, volume = {50}, number = {6}, pages = {3239-3253}, pmid = {35234927}, issn = {1362-4962}, support = {P30 CA093373/CA/NCI NIH HHS/United States ; R21 HG010559/HG/NHGRI NIH HHS/United States ; S10 OD018223/OD/NIH HHS/United States ; }, mesh = {CRISPR-Cas Systems ; Chromatin ; DNA Methylation/genetics ; Epigenesis, Genetic ; *Epigenome ; *Gene Editing/methods ; Gene Silencing ; }, abstract = {Precision epigenome editing has gained significant attention as a method to modulate gene expression without altering genetic information. However, a major limiting factor has been that the gene expression changes are often transient, unlike the life-long epigenetic changes that occur frequently in nature. Here, we systematically interrogate the ability of CRISPR/dCas9-based epigenome editors (Epi-dCas9) to engineer persistent epigenetic silencing. We elucidated cis regulatory features that contribute to the differential stability of epigenetic reprogramming, such as the active transcription histone marks H3K36me3 and H3K27ac strongly correlating with resistance to short-term repression and resistance to long-term silencing, respectively. H3K27ac inversely correlates with increased DNA methylation. Interestingly, the dependance on H3K27ac was only observed when a combination of KRAB-dCas9 and targetable DNA methyltransferases (DNMT3A-dCas9 + DNMT3L) was used, but not when KRAB was replaced with the targetable H3K27 histone methyltransferase Ezh2. In addition, programmable Ezh2/DNMT3A + L treatment demonstrated enhanced engineering of localized DNA methylation and was not sensitive to a divergent chromatin state. Our results highlight the importance of local chromatin features for heritability of programmable silencing and the differential response to KRAB- and Ezh2-based epigenetic editing platforms. The information gained in this study provides fundamental insights into understanding contextual cues to more predictably engineer persistent silencing.}, }
@article {pmid35234622, year = {2022}, author = {Zhang, Z and Hou, W and Chen, S}, title = {Updates on CRISPR-based gene editing in HIV-1/AIDS therapy.}, journal = {Virologica Sinica}, volume = {37}, number = {1}, pages = {1-10}, pmid = {35234622}, issn = {1995-820X}, mesh = {*Acquired Immunodeficiency Syndrome/therapy ; CRISPR-Cas Systems/genetics ; Gene Editing ; Genetic Therapy ; Genome, Human ; *HIV-1/genetics ; Humans ; }, abstract = {Although tremendous efforts have been made to prevent and treat HIV-1 infection, HIV-1/AIDS remains a major threat to global human health. The combination antiretroviral therapy (cART), although able to suppress HIV-1 replication, cannot eliminate the proviral DNA integrated into the human genome and thus requires lifelong treatment that may lead to various side effects. In recent years, clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease 9 (Cas9) related gene-editing systems have been developed and designed as effective ways to treat HIV-1 infection. However, new gene-targeting tools derived from or functioning like CRISPR/Cas9, including base editor, prime editing, SHERLOCK, DETECTR, PAC-MAN, ABACAS, pfAGO, have been developed and optimized for pathogens detection and diseases correction. Here, we summarize recent studies on HIV-1/AIDS gene therapy and provide more gene-editing targets based on studies relating to the molecular mechanism of HIV-1 infection. We also identify the strategies and potential applications of these new gene-editing technologies for HIV-1/AIDS treatment in the future. Moreover, we discuss the caveats and problems that should be addressed before the clinical use of these versatile CRISPR-based gene targeting tools. Finally, we offer alternative solutions to improve the practice of gene targeting in HIV-1/AIDS gene therapy.}, }
@article {pmid35234463, year = {2022}, author = {Wan, Y and Zong, C and Li, X and Wang, A and Li, Y and Yang, T and Bao, Q and Dubow, M and Yang, M and Rodrigo, LA and Mao, C}, title = {New Insights for Biosensing: Lessons from Microbial Defense Systems.}, journal = {Chemical reviews}, volume = {122}, number = {9}, pages = {8126-8180}, doi = {10.1021/acs.chemrev.1c01063}, pmid = {35234463}, issn = {1520-6890}, mesh = {*Biosensing Techniques ; CRISPR-Cas Systems ; *Nanopores ; *Nucleic Acids ; Proteins ; }, abstract = {Microorganisms have gained defense systems during the lengthy process of evolution over millions of years. Such defense systems can protect them from being attacked by invading species (e.g., CRISPR-Cas for establishing adaptive immune systems and nanopore-forming toxins as virulence factors) or enable them to adapt to different conditions (e.g., gas vesicles for achieving buoyancy control). These microorganism defense systems (MDS) have inspired the development of biosensors that have received much attention in a wide range of fields including life science research, food safety, and medical diagnosis. This Review comprehensively analyzes biosensing platforms originating from MDS for sensing and imaging biological analytes. We first describe a basic overview of MDS and MDS-inspired biosensing platforms (e.g., CRISPR-Cas systems, nanopore-forming proteins, and gas vesicles), followed by a critical discussion of their functions and properties. We then discuss several transduction mechanisms (optical, acoustic, magnetic, and electrical) involved in MDS-inspired biosensing. We further detail the applications of the MDS-inspired biosensors to detect a variety of analytes (nucleic acids, peptides, proteins, pathogens, cells, small molecules, and metal ions). In the end, we propose the key challenges and future perspectives in seeking new and improved MDS tools that can potentially lead to breakthrough discoveries in developing a new generation of biosensors with a combination of low cost; high sensitivity, accuracy, and precision; and fast detection. Overall, this Review gives a historical review of MDS, elucidates the principles of emulating MDS to develop biosensors, and analyzes the recent advancements, current challenges, and future trends in this field. It provides a unique critical analysis of emulating MDS to develop robust biosensors and discusses the design of such biosensors using elements found in MDS, showing that emulating MDS is a promising approach to conceptually advancing the design of biosensors.}, }
@article {pmid35234398, year = {2022}, author = {Lu, H and Zhang, Q and Yu, S and Wang, Y and Kang, M and Han, S and Liu, Y and Wang, M}, title = {[Optimization of CRISPR/Cas9-based multiplex base editing in Corynebacterium glutamicum].}, journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology}, volume = {38}, number = {2}, pages = {780-795}, doi = {10.13345/j.cjb.210109}, pmid = {35234398}, issn = {1872-2075}, mesh = {CRISPR-Cas Systems/genetics ; *Corynebacterium glutamicum/genetics/metabolism ; Gene Editing ; Plasmids ; RNA, Guide/genetics/metabolism ; }, abstract = {As a new CRISPR/Cas-derived genome engineering technology, base editing combines the target specificity of CRISPR/Cas and the catalytic activity of nucleobase deaminase to install point mutations at target loci without generating DSBs, requiring exogenous template, or depending on homologous recombination. Recently, researchers have developed a variety of base editing tools in the important industrial strain Corynebacterium glutamicum, and achieved simultaneous editing of two and three genes. However, the multiplex base editing based on CRISPR/Cas9 is still limited by the complexity of multiple sgRNAs, interference of repeated sequence and difficulty of target loci replacement. In this study, multiplex base editing in C. glutamicum was optimized by the following strategies. Firstly, the multiple sgRNA expression cassettes based on individual promoters/terminators was optimized. The target loci can be introduced and replaced rapidly by using a template plasmid and Golden Gate method, which also avoids the interference of repeated sequence. Although the multiple sgRNAs structure is still complicated, the editing efficiency of this strategy is the highest. Then, the multiple gRNA expression cassettes based on Type Ⅱ CRISPR crRNA arrays and tRNA processing were developed. The two strategies only require one single promoter and terminator, and greatly simplify the structure of the expression cassette. Although the editing efficiency has decreased, both methods are still applicable. Taken together, this study provides a powerful addition to the genome editing toolbox of C. glutamicum and facilitates genetic modification of this strain.}, }
@article {pmid35234393, year = {2022}, author = {Yang, Y and Li, N and Zhou, J and Chen, J}, title = {[A CRISPR/dCpf1-based transcriptional repression system for Gluconobacter oxydans].}, journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology}, volume = {38}, number = {2}, pages = {719-736}, doi = {10.13345/j.cjb.210157}, pmid = {35234393}, issn = {1872-2075}, mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; Gene Expression ; *Gluconobacter oxydans/genetics ; Metabolic Engineering ; }, abstract = {Gluconobacter oxydans are widely used in industrial due to its ability of oxidizing carbohydrate rapidly. However, the limited gene manipulation methods and less of efficient gene editing tools impose restrictions on its application in industrial production. In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR) system has been widely used in genome editing and transcriptional regulation which improves the efficiency of genome editing greatly. Here we constructed a CRISPR/dCpf1-mediated gene transcriptional repression system, the expression of a nuclease inactivation Cpf1 protein (dCpf1) in Gluconobacter oxydans together with a 19 nt direct repeats showed effective repression in gene transcription. This system in single gene repression had strong effect and the relative repression level had been increased to 97.9%. While it could be applied in multiplex gene repression which showed strong repression ability at the same time. Furthermore, this system was used in the metabolic pathway of L-sorbose and the regulatory of respiratory chain. The development of CRISPR transcriptional repression system effectively covered the shortage of current gene regulation methods in G. oxydans and provided an efficient gene manipulation tool for metabolic engineering modification in G. oxydans.}, }
@article {pmid35233628, year = {2021}, author = {Bischof, S}, title = {Which factors shape the rice DNA methylome?.}, journal = {The Plant cell}, volume = {33}, number = {9}, pages = {2904-2905}, pmid = {35233628}, issn = {1532-298X}, mesh = {CRISPR-Cas Systems ; DNA ; DNA Methylation/genetics ; *Epigenome ; Methyltransferases/genetics ; *Oryza/genetics ; }, }
@article {pmid35232993, year = {2022}, author = {Rezazade Bazaz, M and Ghahramani Seno, MM and Dehghani, H}, title = {Transposase-CRISPR mediated targeted integration (TransCRISTI) in the human genome.}, journal = {Scientific reports}, volume = {12}, number = {1}, pages = {3390}, pmid = {35232993}, issn = {2045-2322}, mesh = {Animals ; CRISPR-Cas Systems ; Gene Editing/methods ; Gene Knock-In Techniques ; *Genome, Human ; Humans ; Mammals/metabolism ; Plasmids ; *Transposases/genetics/metabolism ; }, abstract = {Various methods have been used in targeted gene knock-in applications. CRISPR-based knock-in strategies based on homology-independent repair pathways such as CRISPR HITI have been shown to possess the best efficiency for gene knock-in in mammalian cells. However, these methods suffer from the probability of plasmid backbone insertion at the target site. On the other hand, studies trying to combine the targeting ability of the Cas9 molecule and the excision/integration capacity of the PB transposase have shown random integrations. In this study, we introduce a new homology-independent knock-in strategy, Transposase-CRISPR mediated Targeted Integration (TransCRISTI), that exploits a fusion of Cas9 nuclease and a double mutant piggyBac transposase. In isogenic mammalian cell lines, we show that the TransCRISTI method demonstrates higher efficiency (72%) for site-specific insertions than the CRISPR HITI (44%) strategy. Application of the TransCRISTI method resulted in site-directed integration in 4.13% and 3.69% of the initially transfected population in the human AAVS1and PML loci, respectively, while the CRISPR HITI strategy resulted in site-directed integration in the PML locus in only 0.6% of cells. We also observed lower off-target and random insertions in the TransCRISTI group than the CRISPR HITI group. The TransCRISTI technology represents a great potential for the accurate and high-efficiency knock-in of the desired transposable elements into the predetermined genomic locations.}, }
@article {pmid35232966, year = {2022}, author = {Schene, IF and Joore, IP and Baijens, JHL and Stevelink, R and Kok, G and Shehata, S and Ilcken, EF and Nieuwenhuis, ECM and Bolhuis, DP and van Rees, RCM and Spelier, SA and van der Doef, HPJ and Beekman, JM and Houwen, RHJ and Nieuwenhuis, EES and Fuchs, SA}, title = {Mutation-specific reporter for optimization and enrichment of prime editing.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {1028}, pmid = {35232966}, issn = {2041-1723}, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genome ; Humans ; Mutation ; RNA, Guide/genetics ; }, abstract = {Prime editing is a versatile genome-editing technique that shows great promise for the generation and repair of patient mutations. However, some genomic sites are difficult to edit and optimal design of prime-editing tools remains elusive. Here we present a fluorescent prime editing and enrichment reporter (fluoPEER), which can be tailored to any genomic target site. This system rapidly and faithfully ranks the efficiency of prime edit guide RNAs (pegRNAs) combined with any prime editor variant. We apply fluoPEER to instruct correction of pathogenic variants in patient cells and find that plasmid editing enriches for genomic editing up to 3-fold compared to conventional enrichment strategies. DNA repair and cell cycle-related genes are enriched in the transcriptome of edited cells. Stalling cells in the G1/S boundary increases prime editing efficiency up to 30%. Together, our results show that fluoPEER can be employed for rapid and efficient correction of patient cells, selection of gene-edited cells, and elucidation of cellular mechanisms needed for successful prime editing.}, }
@article {pmid35232910, year = {2021}, author = {Pan, S and Zhang, H}, title = {Discovery in CRISPR-Cas9 system.}, journal = {Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences}, volume = {46}, number = {12}, pages = {1392-1402}, doi = {10.11817/j.issn.1672-7347.2021.210169}, pmid = {35232910}, issn = {1672-7347}, support = {82073135//the National Natural Science Foundation of China/ ; }, mesh = {Animals ; Bacteria/genetics ; *CRISPR-Cas Systems ; DNA ; Gene Editing ; *Neoplasms/genetics ; }, abstract = {The 2020 Nobel Prize in Chemistry was awarded to the American scientist Jennifer A. Doudna and the French scientist Emmanuelle Charpentier, in recognition of their discovery in one of the greatest weapons in genetic technology: CRISPR-Cas9 gene scissors. The CRISPR-Cas system is a bacterial defense immune system against exogenous genetic material. Because the system can specifically recognize and cut DNA, this technology is widely used for precise editing of animal, plant, and microbial DNA. The discovery of CRISPR-Cas9 gene scissors enables the tedious and complicated cell gene editing work to be completed in a few weeks or even less, which has promoted the development of gene editing technology in various fields and brought revolutionary influence to the field of life sciences. At the same time, CRISPR gene editing technology has become one of the new therapies for tumors because of its large number of targets and relatively simple operation, and it also makes gene therapy possible. Although the technology still needs to solve technical problems such as off-target and promoter inefficiency, the CRISPR-Cas system will show its unique advantages in more fields with the continuous development of life science and basic medicine.}, }
@article {pmid35230602, year = {2022}, author = {Hurley, A and Lagor, WR}, title = {Treating Cardiovascular Disease with Liver Genome Engineering.}, journal = {Current atherosclerosis reports}, volume = {24}, number = {2}, pages = {75-84}, pmid = {35230602}, issn = {1534-6242}, support = {R01 DK124477/DK/NIDDK NIH HHS/United States ; R01 HL132840/HL/NHLBI NIH HHS/United States ; U42 OD026645/OD/NIH HHS/United States ; UG3 HL151545/HL/NHLBI NIH HHS/United States ; }, mesh = {Animals ; *CRISPR-Cas Systems ; *Cardiovascular Diseases/genetics/therapy ; Gene Editing/methods ; Humans ; Liver ; Mice ; }, abstract = {PURPOSE OF REVIEW: This review examines recent progress in somatic genome editing for cardiovascular disease. We briefly highlight new gene editing approaches, delivery systems, and potential targets in the liver.<br><br>RECENT FINDINGS: In recent years, new editing and delivery systems have been applied successfully in model organisms to modify genes within hepatocytes. Disruption of several genes has been shown to dramatically lower plasma cholesterol and triglyceride levels in mice as well as non-human primates. More precise modification of cardiovascular targets has also been achieved through homology-directed repair or base editing. Improved viral vectors and nanoparticle delivery systems are addressing important delivery challenges and helping to mitigate safety concerns. Liver-directed genome editing has the potential to cure both rare and common forms of cardiovascular disease. Exciting progress is already being made, including promising results from preclinical studies and the initiation of human gene therapy trials.}, }
@article {pmid35228726, year = {2022}, author = {Wang, J and He, Z and Wang, G and Zhang, R and Duan, J and Gao, P and Lei, X and Qiu, H and Zhang, C and Zhang, Y and Yin, H}, title = {Efficient targeted insertion of large DNA fragments without DNA donors.}, journal = {Nature methods}, volume = {19}, number = {3}, pages = {331-340}, pmid = {35228726}, issn = {1548-7105}, mesh = {CRISPR-Cas Systems ; DNA/genetics ; *Gene Editing ; Genome ; Genomics ; *RNA, Guide/genetics ; }, abstract = {Targeted insertion of large DNA fragments holds great potential for treating genetic diseases. Prime editors can effectively insert short fragments (~44 bp) but not large ones. Here we developed GRAND editing to precisely insert large DNA fragments without DNA donors. In contrast to prime editors, which require reverse transcription templates hybridizing with the target sequence, GRAND editing employs a pair of prime editing guide RNAs, with reverse transcription templates nonhomologous to the target site but complementary to each other. This strategy exhibited an efficiency of up to 63.0% of a 150-bp insertion with minor by-products and 28.4% of a 250-bp insertion. It allowed insertions up to ~1 kb, although the efficiency remains low for fragments larger than 400 bp. We confirmed efficient insertion in multiple genomic loci of several cell lines and non-dividing cells, which expands the scope of genome editing to enable donor-free insertion of large DNA sequences.}, }
@article {pmid35228123, year = {2022}, author = {Beppu, K and Tsutsumi, R and Ansai, S and Ochiai, N and Terakawa, M and Mori, M and Kuroda, M and Horikawa, K and Tomoi, T and Sakamoto, J and Kamei, Y and Naruse, K and Sakaue, H}, title = {Development of a screening system for agents that modulate taste receptor expression with the CRISPR-Cas9 system in medaka.}, journal = {Biochemical and biophysical research communications}, volume = {601}, number = {}, pages = {65-72}, doi = {10.1016/j.bbrc.2022.02.082}, pmid = {35228123}, issn = {1090-2104}, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Dysgeusia/genetics ; Gene Expression ; Green Fluorescent Proteins/genetics/metabolism ; *Oryzias/genetics ; Quality of Life ; Taste ; }, abstract = {Taste recognition mediated by taste receptors is critical for the survival of animals in nature and is an important determinant of nutritional status and quality of life in humans. However, many factors including aging, diabetes, zinc deficiency, infection with influenza or cold viruses, and chemotherapy can trigger dysgeusia, for which a standard treatment has not been established. We here established an engineered strain of medaka (Oryzias latipes) that expresses green fluorescent protein (GFP) from the endogenous taste 1 receptor 3 (T1R3) gene locus with the use of the CRISPR-Cas9 system. This T1R3-GFP knock-in (KI) strain allows direct visualization of expression from this locus by monitoring of GFP fluorescence. The pattern of GFP expression in the T1R3-GFP KI fish thus mimicked that of endogenous T1R3 gene expression. Furthermore, exposure of T1R3-GFP KI medaka to water containing monosodium glutamate or the anticancer agent 5-fluorouracil resulted in an increase or decrease, respectively, in GFP fluorescence intensity, effects that also recapitulated those on T1R3 mRNA abundance. Finally, screening for agents that affect GFP fluorescence intensity in T1R3-GFP KI medaka identified tryptophan as an amino acid that increases T1R3 gene expression. The establishment of this screening system for taste receptor expression in medaka provides a new tool for the development of potential therapeutic agents for dysgeusia.}, }
@article {pmid35228065, year = {2022}, author = {Najafi, S and Tan, SC and Aghamiri, S and Raee, P and Ebrahimi, Z and Jahromi, ZK and Rahmati, Y and Sadri Nahand, J and Piroozmand, A and Jajarmi, V and Mirzaei, H}, title = {Therapeutic potentials of CRISPR-Cas genome editing technology in human viral infections.}, journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie}, volume = {148}, number = {}, pages = {112743}, doi = {10.1016/j.biopha.2022.112743}, pmid = {35228065}, issn = {1950-6007}, mesh = {COVID-19/therapy ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Genetic Therapy/*methods ; Genome, Viral ; HIV Infections/therapy ; Hepatitis B/therapy ; Herpesviridae Infections/therapy ; Humans ; Papillomavirus Infections/therapy ; SARS-CoV-2 ; Virus Diseases/*therapy ; }, abstract = {Viral infections are a common cause of morbidity worldwide. The emergence of Coronavirus Disease 2019 (COVID-19) has led to more attention to viral infections and finding novel therapeutics. The CRISPR-Cas9 system has been recently proposed as a potential therapeutic tool for the treatment of viral diseases. Here, we review the research progress in the use of CRISPR-Cas technology for treating viral infections, as well as the strategies for improving the delivery of this gene-editing tool in vivo. Key challenges that hinder the widespread clinical application of CRISPR-Cas9 technology are also discussed, and several possible directions for future research are proposed.}, }
@article {pmid35227160, year = {2022}, author = {Chattopadhyay, I and J, RB and Usman, TMM and Varjani, S}, title = {Exploring the role of microbial biofilm for industrial effluents treatment.}, journal = {Bioengineered}, volume = {13}, number = {3}, pages = {6420-6440}, pmid = {35227160}, issn = {2165-5987}, mesh = {*Biodegradation, Environmental ; *Biofilms ; CRISPR-Cas Systems ; Gene Editing ; Genome, Bacterial/genetics ; *Industrial Waste ; *Quorum Sensing ; *Waste Water ; }, abstract = {Biofilm formation on biotic or abiotic surfaces is caused by microbial cells of a single or heterogeneous species. Biofilm protects microbes from stressful environmental conditions, toxic action of chemicals, and antimicrobial substances. Quorum sensing (QS) is the generation of autoinducers (AIs) by bacteria in a biofilm to communicate with one other. QS is responsible for the growth of biofilm, synthesis of exopolysaccharides (EPS), and bioremediation of environmental pollutants. EPS is used for wastewater treatment due to its three-dimensional matrix which is composed of proteins, polysaccharides, humic-like substances, and nucleic acids. Autoinducers mediate significantly the degradation of environmental pollutants. Acyl-homoserine lactone (AHL) producing bacteria as well as quorum quenching enzyme or bacteria can effectively improve the performance of wastewater treatment. Biofilms-based reactors due to their economic and ecofriendly nature are used for the treatment of industrial wastewaters. Electrodes coated with electro-active biofilm (EAB) which are obtained from sewage sludge, activated sludge, or industrial and domestic effluents are getting popularity in bioremediation. Microbial fuel cells are involved in wastewater treatment and production of energy from wastewater. Synthetic biological systems such as genome editing by CRISPR-Cas can be used for the advanced bioremediation process through modification of metabolic pathways in quorum sensing within microbial communities. This narrative review discusses the impacts of QS regulatory approaches on biofilm formation, extracellular polymeric substance synthesis, and role of microbial community in bioremediation of pollutants from industrial effluents.}, }
@article {pmid35225758, year = {2022}, author = {Jin, CZ and Jin, L and Liu, MJ and Kang, MK and Park, SH and Park, DJ and Kim, CJ}, title = {Salinarimonas soli sp. nov., isolated from soil.}, journal = {International journal of systematic and evolutionary microbiology}, volume = {72}, number = {2}, pages = {}, doi = {10.1099/ijsem.0.005095}, pmid = {35225758}, issn = {1466-5034}, mesh = {Alphaproteobacteria/*classification/isolation &amp; purification ; Bacterial Typing Techniques ; Base Composition ; DNA, Bacterial/genetics ; Fatty Acids/chemistry ; Phospholipids/chemistry ; *Phylogeny ; RNA, Ribosomal, 16S/genetics ; Republic of Korea ; Sequence Analysis, DNA ; *Soil Microbiology ; Ubiquinone/analogs &amp; derivatives/chemistry ; }, abstract = {A light pink coloured bacterium, designated strain BN140002T, was isolated from a soil sample collected in Goesan-gun, Chungcheongbuk-do, Republic of Korea. Cells of strain BN140002T were Gram-stain-negative, aerobic, motile and rod-shaped. Phylogenetic analysis based on 16S rRNA gene sequences showed 94.7, 94.7, 93.9, 93.3, 93.4 and 93.0% similarities to Salinarimonas rosea KCTC 22346T, Salinarimonas ramus DSM 22962T, Saliniramus fredricksonii HL-109T, Microvirga soli R491T, Chelatococcus caeni EBR-4-1T and Chelatococcus composti PC-2T, respectively. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine and phosphatidylethanolamine. The major cellular fatty acids were summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and summed feature 1 (C12 : 0 aldehyde and/or unknown 10.98) and the predominant ubiquinone was Q-10. The genomic DNA G+C content of strain BN140002T was 70.1 mol%. The genomic orthoANI values between strain BN140002T and Salinarimonas rosea KCTC 22346T and Salinarimonas ramus DSM 22962T were 75.0 and 74.8 %, respectively. Strain BN140002T had a class I-C type CRISPR-Cas system (CRISPR-associated helicase Cas3, CRISPR-associated protein Cas8c, CRISPR-associated protein Cas7, CRISPR-associated RecB family exonuclease Cas4, CRISPR-associated protein 1, 2). Based on phenotypic, chemotaxonomic and phylogenetic data, strain BN140002T should be assigned as a novel species of the genus Salinarimonas, for which the name Salinarimonas soli sp. nov. is proposed. The type strain is BN140002T (=KCTC 42643T=CCTCC AB 2017173T).}, }
@article {pmid35225591, year = {2022}, author = {Kotikam, V and Gajula, PK and Coyle, L and Rozners, E}, title = {Amide Internucleoside Linkages Are Well Tolerated in Protospacer Adjacent Motif-Distal Region of CRISPR RNAs.}, journal = {ACS chemical biology}, volume = {17}, number = {3}, pages = {509-512}, doi = {10.1021/acschembio.1c00900}, pmid = {35225591}, issn = {1554-8937}, support = {R35 GM130207/GM/NIGMS NIH HHS/United States ; }, mesh = {*Amides/chemistry ; CRISPR-Cas Systems/genetics ; DNA Cleavage ; *Gene Editing/methods ; RNA Interference ; RNA, Small Interfering/chemistry ; }, abstract = {The development of CRISPR-Cas9 mediated gene editing technology is revolutionizing molecular biology, biotechnology, and medicine. However, as with other nucleic acid technologies, CRISPR would greatly benefit from chemical modifications that optimize delivery, activity, and specificity of gene editing. Amide modifications at certain positions of short interfering RNAs have been previously shown to improve their RNAi activity and specificity, which motivated the current study on replacement of selected internucleoside phosphates of CRISPR RNAs with amide linkages. Herein, we show that amide modifications did not interfere with CRISPR-Cas9 activity when placed in the protospacer adjacent motif (PAM) distal region of CRISPR RNAs. In contrast, modification of the seed region led to a loss of DNA cleavage activity at most but not all positions. These results are encouraging for future studies on amides as backbone modifications in CRISPR RNAs.}, }
@article {pmid35225354, year = {2022}, author = {Sun, Y and Zhang, Y and Zhang, D and Wang, G and Song, L and Liu, Z}, title = {In vivo CRISPR-Cas9-mediated DNA chop identifies a cochlear outer hair cell-specific enhancer.}, journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology}, volume = {36}, number = {4}, pages = {e22233}, doi = {10.1096/fj.202100421RR}, pmid = {35225354}, issn = {1530-6860}, mesh = {Animals ; *CRISPR-Cas Systems ; Cochlea/*metabolism ; *Enhancer Elements, Genetic ; Female ; Green Fluorescent Proteins/genetics/metabolism ; Hair Cells, Auditory, Outer/*metabolism ; Humans ; *Introns ; Male ; Mice ; Mice, Transgenic ; Molecular Motor Proteins/genetics/*metabolism ; Sulfate Transporters/antagonists &amp; inhibitors/*genetics/metabolism ; }, abstract = {Cochlear outer hair cells (OHCs) are essential for hearing. A short, OHC-specific enhancer is necessary but not yet available for gene therapeutic applications in OHC damage. Such damage is a major cause of deafness. Prestin is a motor protein exclusively expressed in OHCs. We hypothesized that the cis-regulatory DNA fragment deletion of Slc26a5 would affect its expression. We tested this hypothesis by conducting CRISPR/Cas9-mediated large DNA fragment deletion of mouse Slc26a5 intron regions. First, starting from a ~13 kbp fragment, step-by-step, we narrowed down the sequence to a 1.4 kbp segment. By deleting either a 13 kbp or 1.4 kbp fragment, we observed delayed Prestin expression. Second, we showed that 1.4 kbp was an OHC-specific enhancer because enhanced green fluorescent protein (EGFP) was highly and specifically expressed in OHCs in a transgenic mouse where EGFP was driven by the 1.4 kbp segment. More importantly, specific EGFP was also driven by its homologous 398 bp fragment in human Slc26a5. This suggests that the enhancer is likely to be evolutionarily conserved across different species.}, }
@article {pmid35225345, year = {2022}, author = {Puchta, H and Jiang, J and Wang, K and Zhao, Y}, title = {Updates on gene editing and its applications.}, journal = {Plant physiology}, volume = {188}, number = {4}, pages = {1725-1730}, pmid = {35225345}, issn = {1532-2548}, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; }, }
@article {pmid35224232, year = {2022}, author = {Huang, C and Wang, C and Luo, Y}, title = {Research progress of pathway and genome evolution in microbes.}, journal = {Synthetic and systems biotechnology}, volume = {7}, number = {1}, pages = {648-656}, pmid = {35224232}, issn = {2405-805X}, abstract = {Microbes can produce valuable natural products widely applied in medicine, food and other important fields. Nevertheless, it is usually challenging to achieve ideal industrial yields due to low production rate and poor toxicity tolerance. Evolution is a constant mutation and adaptation process used to improve strain performance. Generally speaking, the synthesis of natural products in microbes is often intricate, involving multiple enzymes or multiple pathways. Individual evolution of a certain enzyme often fails to achieve the desired results, and may lead to new rate-limiting nodes that affect the growth of microbes. Therefore, it is inevitable to evolve the biosynthetic pathways or the whole genome. Here, we reviewed the pathway-level evolution including multi-enzyme evolution, regulatory elements engineering, and computer-aided engineering, as well as the genome-level evolution based on several tools, such as genome shuffling and CRISPR/Cas systems. Finally, we also discussed the major challenges faced by in vivo evolution strategies and proposed some potential solutions.}, }
@article {pmid35222448, year = {2021}, author = {Nguyen, DV and Hoang, TT and Le, NT and Tran, HT and Nguyen, CX and Moon, YH and Chu, HH and Do, PT}, title = {An Efficient Hairy Root System for Validation of Plant Transformation Vector and CRISPR/Cas Construct Activities in Cucumber (Cucumis sativus L.).}, journal = {Frontiers in plant science}, volume = {12}, number = {}, pages = {770062}, pmid = {35222448}, issn = {1664-462X}, abstract = {Hairy root induction system has been applied in various plant species as an effective method to study gene expression and function due to its fast-growing and high genetic stability. Recently, these systems have shown to be an effective tool to evaluate activities of CRISPR/Cas9 systems for genome editing. In this study, Rhizobium rhizogenes mediated hairy root induction was optimized to provide an effective tool for validation of plant transformation vector, CRISPR/Cas9 construct activities as well as selection of targeted gRNAs for gene editing in cucumber (Cucumis sativus L.). Under the optimized conditions including OD650 at 0.4 for infection and 5 days of co-cultivation, the highest hairy root induction frequency reached 100% for the cucumber variety Choka F1. This procedure was successfully utilized to overexpress a reporter gene (gus) and induce mutations in two Lotus japonicus ROOTHAIRLESS1 homolog genes CsbHLH66 and CsbHLH82 using CRISPR/Cas9 system. For induced mutation, about 78% of transgenic hairy roots exhibited mutant phenotypes including sparse root hair and root hair-less. The targeted mutations were obtained in individual CsbHLH66, CsbHLH82, or both CsbHLH66 and CsbHLH82 genes by heteroduplex analysis and sequencing. The hairy root transformation system established in this study is sufficient and potential for further research in genome editing of cucumber as well as other cucumis plants.}, }
@article {pmid35222299, year = {2021}, author = {Wentz, TG and Tremblay, BJM and Bradshaw, M and Doxey, AC and Sharma, SK and Sauer, JD and Pellett, S}, title = {Endogenous CRISPR-Cas Systems in Group I Clostridium botulinum and Clostridium sporogenes Do Not Directly Target the Botulinum Neurotoxin Gene Cluster.}, journal = {Frontiers in microbiology}, volume = {12}, number = {}, pages = {787726}, pmid = {35222299}, issn = {1664-302X}, support = {R21 AI144060/AI/NIAID NIH HHS/United States ; }, abstract = {Most strains of proteolytic group I Clostridium botulinum (G1 C. botulinum) and some strains of Clostridium sporogenes possess genes encoding botulinum neurotoxin (BoNT), a potent neuroparalytic agent. Within G1 C. botulinum, conserved bont gene clusters of three major toxin serotypes (bont/A/B/F) can be found on conjugative plasmids and/or within chromosomal pathogenicity islands. CRISPR-Cas systems enable site-specific targeting of previously encountered mobile genetic elements (MGE) such as plasmids and bacteriophage through the creation of a spacer library complementary to protospacers within the MGEs. To examine whether endogenous CRISPR-Cas systems restrict the transfer of bont gene clusters across strains we conducted a bioinformatic analysis profiling endogenous CRISPR-Cas systems from 241 G1 C. botulinum and C. sporogenes strains. Approximately 6,200 CRISPR spacers were identified across the strains and Type I-B, III-A/B/D cas genes and CRISPR array features were identified in 83% of the strains. Mapping the predicted spacers against the masked strain and RefSeq plasmid dataset identified 56,000 spacer-protospacer matches. While spacers mapped heavily to targets within bont(+) plasmids, no protospacers were identified within the bont gene clusters. These results indicate the toxin is not a direct target of CRISPR-Cas but the plasmids predominantly responsible for its mobilization are. Finally, while the presence of a CRISPR-Cas system did not reliably indicate the presence or absence of a bont gene cluster, comparative genomics across strains indicates they often occupy the same hypervariable loci common to both species, potentially suggesting similar mechanisms are involved in the acquisition and curation of both genomic features.}, }
@article {pmid35219699, year = {2022}, author = {Ganguly, K and Cox, JL and Ghersi, D and Grandgenett, PM and Hollingsworth, MA and Jain, M and Kumar, S and Batra, SK}, title = {Mucin 5AC-Mediated CD44/ITGB1 Clustering Mobilizes Adipose-Derived Mesenchymal Stem Cells to Modulate Pancreatic Cancer Stromal Heterogeneity.}, journal = {Gastroenterology}, volume = {162}, number = {7}, pages = {2032-2046.e12}, doi = {10.1053/j.gastro.2022.02.032}, pmid = {35219699}, issn = {1528-0012}, support = {R01 CA206444/CA/NCI NIH HHS/United States ; U01 CA200466/CA/NCI NIH HHS/United States ; R01 CA247471/CA/NCI NIH HHS/United States ; R44 CA235991/CA/NCI NIH HHS/United States ; R01 CA183459/CA/NCI NIH HHS/United States ; R01 CA210637/CA/NCI NIH HHS/United States ; P01 CA217798/CA/NCI NIH HHS/United States ; }, mesh = {Actins/metabolism ; Animals ; Cluster Analysis ; Humans ; Hyaluronan Receptors/metabolism ; *Mesenchymal Stem Cells/metabolism ; Mice ; Mucin 5AC/metabolism ; *Pancreatic Neoplasms/genetics/metabolism/therapy ; Proto-Oncogene Proteins p21(ras)/genetics/metabolism ; }, abstract = {BACKGROUND &amp; AIMS: Secreted mucin 5AC (MUC5AC) promotes pancreatic cancer (PC) progression and chemoresistance, suggesting its clinical association with poor prognosis. RNA sequencing analysis from the autochthonous pancreatic tumors showed a significant stromal alteration on genetic ablation of Muc5ac. Previously, depletion or targeting the stromal fibroblasts showed an ambiguous effect on PC pathogenesis. Hence, identifying the molecular players and mechanisms driving fibroblast heterogeneity is critical for improved clinical outcomes.<br><br>METHODS: Autochthonous murine models of PC (KrasG12D, Pdx1-Cre [KC] and KrasG12D, Pdx1-Cre, Muc5ac-/- [KCM]) and co-implanted allografts of murine PC cell lines (Muc5ac wild-type and CRISPR/Cas knockout) with adipose-derived mesenchymal stem cells (AD-MSCs) were used to assess the role of Muc5ac in stromal heterogeneity. Proliferation, migration, and surface expression of cell-adhesion markers on AD-MSCs were measured using live-cell imaging and flow cytometry. MUC5AC-interactome was investigated using mass-spectrometry and enzyme-linked immunosorbent assay.<br><br>RESULTS: The KCM tumors showed a significant decrease in the expression of α-smooth muscle actin and fibronectin compared with histology-matched KC tumors. Our study showed that MUC5AC, carrying tumor secretome, gets enriched in the adipose tissues of tumor-bearing mice and patients with PC, promoting CD44/CD29 (integrin-β1) clustering that leads to Rac1 activation and migration of AD-MSCs. Furthermore, treatment with KC-derived serum enhanced proliferation and migration of AD-MSCs, which was abolished on Muc5ac-depletion or pharmacologic inhibition of CXCR2 and Rac1, respectively. The AD-MSCs significantly contribute toward α-smooth muscle actin-positive cancer-associated fibroblasts population in Muc5ac-dependent manner, as suggested by autochthonous tumors, co-implantation xenografts, and patient tumors.<br><br>CONCLUSION: MUC5AC, secreted during PC progression, enriches in adipose and enhances the mobilization of AD-MSCs. On recruitment to pancreatic tumors, AD-MSCs proliferate and contribute towards stromal heterogeneity.}, }
@article {pmid35219382, year = {2022}, author = {Tsuchida, CA and Zhang, S and Doost, MS and Zhao, Y and Wang, J and O'Brien, E and Fang, H and Li, CP and Li, D and Hai, ZY and Chuck, J and Brötzmann, J and Vartoumian, A and Burstein, D and Chen, XW and Nogales, E and Doudna, JA and Liu, JG}, title = {Chimeric CRISPR-CasX enzymes and guide RNAs for improved genome editing activity.}, journal = {Molecular cell}, volume = {82}, number = {6}, pages = {1199-1209.e6}, doi = {10.1016/j.molcel.2022.02.002}, pmid = {35219382}, issn = {1097-4164}, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Endonucleases/genetics/metabolism ; *Gene Editing/methods ; Mammals/metabolism ; RNA/genetics ; *RNA, Guide/genetics/metabolism ; }, abstract = {A compact protein with a size of <1,000 amino acids, the CRISPR-associated protein CasX is a fundamentally distinct RNA-guided nuclease when compared to Cas9 and Cas12a. Although it can induce RNA-guided genome editing in mammalian cells, the activity of CasX is less robust than that of the widely used S. pyogenes Cas9. Here, we show that structural features of two CasX homologs and their guide RNAs affect the R-loop complex assembly and DNA cleavage activity. Cryo-EM-based structural engineering of either the CasX protein or the guide RNA produced two new CasX genome editors (DpbCasX-R3-v2 and PlmCasX-R1-v2) with significantly improved DNA manipulation efficacy. These results advance both the mechanistic understanding of CasX and its application as a genome-editing tool.}, }
@article {pmid35219341, year = {2022}, author = {Stovicek, V and Dato, L and Almqvist, H and Schöpping, M and Chekina, K and Pedersen, LE and Koza, A and Figueira, D and Tjosås, F and Ferreira, BS and Forster, J and Lidén, G and Borodina, I}, title = {Rational and evolutionary engineering of Saccharomyces cerevisiae for production of dicarboxylic acids from lignocellulosic biomass and exploring genetic mechanisms of the yeast tolerance to the biomass hydrolysate.}, journal = {Biotechnology for biofuels and bioproducts}, volume = {15}, number = {1}, pages = {22}, pmid = {35219341}, issn = {2731-3654}, support = {FP7-613771//Seventh Framework Programme/ ; NNF20OC0060809//Novo Nordisk Fonden/ ; NNF20CC0035580//Novo Nordisk Fonden/ ; 757384//H2020 European Research Council/ ; }, abstract = {BACKGROUND: Lignosulfonates are significant wood chemicals with a $700 million market, produced by sulfite pulping of wood. During the pulping process, spent sulfite liquor (SSL) is generated, which in addition to lignosulfonates contains hemicellulose-derived sugars-in case of hardwoods primarily the pentose sugar xylose. The pentoses are currently underutilized. If they could be converted into value-added chemicals, overall economic profitability of the process would increase. SSLs are typically very inhibitory to microorganisms, which presents a challenge for a biotechnological process. The aim of the present work was to develop a robust yeast strain able to convert xylose in SSL to carboxylic acids.<br><br>RESULTS: The industrial strain Ethanol Red of the yeast Saccharomyces cerevisiae was engineered for efficient utilization of xylose in a Eucalyptus globulus lignosulfonate stream at low pH using CRISPR/Cas genome editing and adaptive laboratory evolution. The engineered strain grew in synthetic medium with xylose as sole carbon source with maximum specific growth rate (µmax) of 0.28 1/h. Selected evolved strains utilized all carbon sources in the SSL at pH 3.5 and grew with µmax between 0.05 and 0.1 1/h depending on a nitrogen source supplement. Putative genetic determinants of the increased tolerance to the SSL were revealed by whole genome sequencing of the evolved strains. In particular, four top-candidate genes (SNG1, FIT3, FZF1 and CBP3) were identified along with other gene candidates with predicted important roles, based on the type and distribution of the mutations across different strains and especially the best performing ones. The developed strains were further engineered for production of dicarboxylic acids (succinic and malic acid) via overexpression of the reductive branch of the tricarboxylic acid cycle (TCA). The production strain produced 0.2 mol and 0.12 mol of malic acid and succinic acid, respectively, per mol of xylose present in the SSL.<br><br>CONCLUSIONS: The combined metabolic engineering and adaptive evolution approach provided a robust SSL-tolerant industrial strain that converts fermentable carbon content of the SSL feedstock into malic and succinic acids at low pH.in production yields reaching 0.1 mol and 0.065 mol per mol of total consumed carbon sources.. Moreover, our work suggests potential genetic background of the tolerance to the SSL stream pointing out potential gene targets for improving the tolerance to inhibitory industrial feedstocks.}, }
@article {pmid35217837, year = {2022}, author = {Mallapaty, S}, title = {How to protect the first 'CRISPR babies' prompts ethical debate.}, journal = {Nature}, volume = {603}, number = {7900}, pages = {213-214}, pmid = {35217837}, issn = {1476-4687}, mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; Morals ; }, }
@article {pmid35217666, year = {2022}, author = {Toral, MA and Charlesworth, CT and Ng, B and Chemudupati, T and Homma, S and Nakauchi, H and Bassuk, AG and Porteus, MH and Mahajan, VB}, title = {Investigation of Cas9 antibodies in the human eye.}, journal = {Nature communications}, volume = {13}, number = {1}, pages = {1053}, pmid = {35217666}, issn = {2041-1723}, support = {P30 EY026877/EY/NEI NIH HHS/United States ; T32 GM139776/GM/NIGMS NIH HHS/United States ; R01 EY030151/EY/NEI NIH HHS/United States ; R01 EY024698/EY/NEI NIH HHS/United States ; T32 GM007337/GM/NIGMS NIH HHS/United States ; R01 EY031952/EY/NEI NIH HHS/United States ; R01 EY025225/EY/NEI NIH HHS/United States ; }, mesh = {Animals ; Antibodies/metabolism ; *CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems ; Humans ; Mice ; Streptococcus pyogenes/metabolism ; T-Lymphocytes ; }, abstract = {Preexisting immunity against Cas9 proteins in humans represents a safety risk for CRISPR-Cas9 technologies. However, it is unclear to what extent preexisting Cas9 immunity is relevant to the eye as it is targeted for early in vivo CRISPR-Cas9 clinical trials. While the eye lacks T-cells, it contains antibodies, cytokines, and resident immune cells. Although precise mechanisms are unclear, intraocular inflammation remains a major cause of vision loss. Here, we used immunoglobulin isotyping and ELISA platforms to profile antibodies in serum and vitreous fluid biopsies from human adult subjects and Cas9-immunized mice. We observed high prevalence of preexisting Cas9-reactive antibodies in serum but not in the eye. However, we detected intraocular antibodies reactive to S. pyogenes-derived Cas9 after S. pyogenes intraocular infection. Our data suggest that serum antibody concentration may determine whether specific intraocular antibodies develop, but preexisting immunity to Cas9 may represent a lower risk in human eyes than systemically.}, }
@article {pmid35217603, year = {2022}, author = {Petersen, GEL and Buntjer, JB and Hely, FS and Byrne, TJ and Doeschl-Wilson, A}, title = {Modeling suggests gene editing combined with vaccination could eliminate a persistent disease in livestock.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {119}, number = {9}, pages = {}, pmid = {35217603}, issn = {1091-6490}, mesh = {Animals ; CRISPR-Cas Systems ; *Gene Editing ; Livestock/*genetics ; Porcine Reproductive and Respiratory Syndrome/*prevention &amp; control ; Porcine respiratory and reproductive syndrome virus/*genetics/immunology ; Proof of Concept Study ; Swine ; *Vaccination ; }, abstract = {Recent breakthroughs in gene-editing technologies that can render individual animals fully resistant to infections may offer unprecedented opportunities for controlling future epidemics in farm animals. Yet, their potential for reducing disease spread is poorly understood as the necessary theoretical framework for estimating epidemiological effects arising from gene-editing applications is currently lacking. Here, we develop semistochastic modeling approaches to investigate how the adoption of gene editing may affect infectious disease prevalence in farmed animal populations and the prospects and time scale for disease elimination. We apply our models to the porcine reproductive and respiratory syndrome (PRRS), one of the most persistent global livestock diseases to date. Whereas extensive control efforts have shown limited success, recent production of gene-edited pigs that are fully resistant to the PRRS virus have raised expectations for eliminating this deadly disease. Our models predict that disease elimination on a national scale would be difficult to achieve if gene editing was used as the only disease control. However, from a purely epidemiological perspective, disease elimination may be achievable within 3 to 6 y, if gene editing were complemented with widespread and sufficiently effective vaccination. Besides strategic distribution of genetically resistant animals, several other key determinants underpinning the epidemiological impact of gene editing were identified.}, }
@article {pmid35217600, year = {2022}, author = {Kwon, T and Ra, JS and Lee, S and Baek, IJ and Khim, KW and Lee, EA and Song, EK and Otarbayev, D and Jung, W and Park, YH and Wie, M and Bae, J and Cheng, H and Park, JH and Kim, N and Seo, Y and Yun, S and Kim, HE and Moon, HE and Paek, SH and Park, TJ and Park, YU and Rhee, H and Choi, JH and Cho, SW and Myung, K}, title = {Precision targeting tumor cells using cancer-specific InDel mutations with CRISPR-Cas9.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {119}, number = {9}, pages = {}, pmid = {35217600}, issn = {1091-6490}, mesh = {Animals ; *CRISPR-Cas Systems ; Cell Death/genetics ; DNA Breaks, Double-Stranded ; Heterografts ; Humans ; *INDEL Mutation ; Mice ; Neoplasms/*genetics ; }, abstract = {An ideal cancer therapeutic strategy involves the selective killing of cancer cells without affecting the surrounding normal cells. However, researchers have failed to develop such methods for achieving selective cancer cell death because of shared features between cancerous and normal cells. In this study, we have developed a therapeutic strategy called the cancer-specific insertions-deletions (InDels) attacker (CINDELA) to selectively induce cancer cell death using the CRISPR-Cas system. CINDELA utilizes a previously unexplored idea of introducing CRISPR-mediated DNA double-strand breaks (DSBs) in a cancer-specific fashion to facilitate specific cell death. In particular, CINDELA targets multiple InDels with CRISPR-Cas9 to produce many DNA DSBs that result in cancer-specific cell death. As a proof of concept, we demonstrate here that CINDELA selectively kills human cancer cell lines, xenograft human tumors in mice, patient-derived glioblastoma, and lung patient-driven xenograft tumors without affecting healthy human cells or altering mouse growth.}, }
@article {pmid35217495, year = {2022}, author = {Zhou, H and Sun, H and Rong, Z and Cui, W}, title = {Generation of Mt3 Homozygote murine ES cell lines via CRISPR/Cas9 technology.}, journal = {Stem cell research}, volume = {60}, number = {}, pages = {102714}, doi = {10.1016/j.scr.2022.102714}, pmid = {35217495}, issn = {1876-7753}, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Cell Line ; Homozygote ; Mice ; Phenotype ; *Technology ; }, abstract = {Metallothioneins belong to a superfamily of intracellular metal-binding proteins, have bearing on almost all biochemical processes,include free radical scavenging, apoptosis, regulation of intracellular redox balance, and anti-inflammatory processes. By using a CRISPR/Cas 9 system, we generated Mt3-/- mES line. This cell line has contributed to further investigation of the functions of Mtt3 during early development, as well as a cell model for screening for metal-related detoxificationan and antioxidant response phenotype during stem cell differentiation.}, }
@article {pmid35217482, year = {2022}, author = {Son, JS and Park, CY and Lee, G and Park, JY and Kim, HJ and Kim, G and Chi, KY and Woo, DH and Han, C and Kim, SK and Park, HJ and Kim, DW and Kim, JH}, title = {Therapeutic correction of hemophilia A using 2D endothelial cells and multicellular 3D organoids derived from CRISPR/Cas9-engineered patient iPSCs.}, journal = {Biomaterials}, volume = {283}, number = {}, pages = {121429}, doi = {10.1016/j.biomaterials.2022.121429}, pmid = {35217482}, issn = {1878-5905}, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Endothelial Cells/metabolism ; *Hemophilia A/genetics/metabolism/therapy ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; Mice ; Organoids/metabolism ; }, abstract = {The bleeding disorder hemophilia A (HA) is caused by a single-gene (F8) defect and its clinical symptom can be substantially improved by a small increase in the plasma coagulation factor VIII (FVIII) level. In this study, we used F8-defective human induced pluripotent stem cells from an HA patient (F8d-HA hiPSCs) and F8-corrected (F8c) HA hiPSCs produced by CRISPR/Cas9 genome engineering of F8d-HA hiPSCs. We obtained a highly enriched population of CD157+ cells from CRISPR/Cas9-edited F8c-HA hiPSCs. These cells exhibited multiple cellular and functional phenotypes of endothelial cells (ECs) with significant levels of FVIII activity, which was not observed in F8d-HA hiPSC-ECs. After transplantation, the engineered F8c-HA hiPSC-ECs dramatically changed bleeding episodes in HA animals and restored plasma FVIII activity. Notably, grafting a high dose of ECs substantially reduced the bleeding time during multiple consecutive bleeding challenges in HA mice, demonstrating a robust hemostatic effect (90% survival). Furthermore, the engrafted ECs survived more than 3 months in HA mice and reversed bleeding phenotypes against lethal wounding challenges. We also produced F8c-HA hiPSC-derived 3D liver organoids by assembling three different cell types in microwell devices and confirmed its therapeutic effect in HA animals. Our data demonstrate that the combination of genome-engineering and iPSC technologies represents a novel modality that allows autologous cell-mediated gene therapy for treating HA.}, }
@article {pmid35217295, year = {2022}, author = {Dong, C and Gou, Y and Lian, J}, title = {SgRNA engineering for improved genome editing and expanded functional assays.}, journal = {Current opinion in biotechnology}, volume = {75}, number = {}, pages = {102697}, doi = {10.1016/j.copbio.2022.102697}, pmid = {35217295}, issn = {1879-0429}, abstract = {The CRISPR/Cas system has been established as the most powerful and practical genome engineering tool for both fundamental researches and biotechnological applications. Great efforts have been devoted to engineering the CRISPR system with better performance and novel functions. As an essential component, single guide RNAs (sgRNAs) have been extensively designed and engineered with desirable functions. This review highlights representative studies that optimize the sgRNA nucleotide sequences for improved genome editing performance (e.g. activity and specificity) as well as add extra aptamers and end extensions for expanded CRISPR-based functional assays (e.g. transcriptional regulation, genome imaging, and prime editor). The perspectives for further sgRNA engineering to establish more powerful and versatile CRISPR/Cas systems are also discussed.}, }
@article {pmid35216669, year = {2022}, author = {Wimmer, F and Mougiakos, I and Englert, F and Beisel, CL}, title = {Rapid cell-free characterization of multi-subunit CRISPR effectors and transposons.}, journal = {Molecular cell}, volume = {82}, number = {6}, pages = {1210-1224.e6}, doi = {10.1016/j.molcel.2022.01.026}, pmid = {35216669}, issn = {1097-4164}, mesh = {*CRISPR-Cas Systems ; Cell-Free System/metabolism ; DNA/genetics ; *Endonucleases/genetics ; RNA/metabolism ; }, abstract = {CRISPR-Cas biology and technologies have been largely shaped to date by the characterization and use of single-effector nucleases. By contrast, multi-subunit effectors dominate natural systems, represent emerging technologies, and were recently associated with RNA-guided DNA transposition. This disconnect stems from the challenge of working with multiple protein subunits in vitro and in vivo. Here, we apply cell-free transcription-translation (TXTL) systems to radically accelerate the characterization of multi-subunit CRISPR effectors and transposons. Numerous DNA constructs can be combined in one TXTL reaction, yielding defined biomolecular readouts in hours. Using TXTL, we mined phylogenetically diverse I-E effectors, interrogated extensively self-targeting I-C and I-F systems, and elucidated targeting rules for I-B and I-F CRISPR transposons using only DNA-binding components. We further recapitulated DNA transposition in TXTL, which helped reveal a distinct branch of I-B CRISPR transposons. These capabilities will facilitate the study and exploitation of the broad yet underexplored diversity of CRISPR-Cas systems and transposons.}, }
@article {pmid35216463, year = {2022}, author = {Wu, Q and Liu, Y and Huang, J}, title = {CRISPR-Cas9 Mediated Mutation in OsPUB43 Improves Grain Length and Weight in Rice by Promoting Cell Proliferation in Spikelet Hull.}, journal = {International journal of molecular sciences}, volume = {23}, number = {4}, pages = {}, pmid = {35216463}, issn = {1422-0067}, support = {cstc2020jcyj-msxmX0656//Natural Science Foundation of Chongqing/ ; 2020CDJ-LHZZ-034//Fundamental Research Funds for the Central Universities/ ; }, mesh = {CRISPR-Cas Systems ; *Cell Proliferation ; Edible Grain/*anatomy &amp; histology/physiology ; Gene Editing ; *Mutation ; Oryza/anatomy &amp; histology/*enzymology/metabolism/physiology ; Plant Proteins/genetics ; Ubiquitin-Protein Ligases/*genetics ; }, abstract = {Grain weight, a crucial trait that determines the grain yield in rice, is influenced by grain size. Although a series of regulators that control grain size have been identified in rice, the mechanisms underlying grain development are not yet well understood. In this study, we identified OsPUB43, a U-box E3 ubiquitin ligase, as an important negative regulator determining the gain size and grain weight in rice. Phenotypes of large grain are observed in ospub43 mutants, whereas overexpression of OsPUB43 results in short grains. Scanning electron microscopy analysis reveals that OsPUB43 modulates the grain size mainly by inhibiting cell proliferation in the spikelet hull. The OsPUB43 protein is localized in the cytoplasm and nucleus. The ospub43 mutants display high sensitivity to exogenous BR, while OsPUB43-OE lines are hyposensitive to BR. Furthermore, the transient transcriptional activity assay shows that OsBZR1 can activate the expression of OsPUB43. Collectively, our results indicate that OsPUB43 negatively controls the gain size by modulating the expression of BR-responsive genes as well as MADS-box genes that are required for lemma/palea specification, suggesting that OsPUB43 has a potential valuable application in the enlargement of grain size in rice.}, }
@article {pmid35216449, year = {2022}, author = {Simbulan-Rosenthal, CM and Haribabu, Y and Vakili, S and Kuo, LW and Clark, H and Dougherty, R and Alobaidi, R and Carney, B and Sykora, P and Rosenthal, DS}, title = {Employing CRISPR-Cas9 to Generate CD133 Synthetic Lethal Melanoma Stem Cells.}, journal = {International journal of molecular sciences}, volume = {23}, number = {4}, pages = {}, pmid = {35216449}, issn = {1422-0067}, support = {5 R42 ES026908 03/ES/NIEHS NIH HHS/United States ; 1R41ES032435-01/ES/NIEHS NIH HHS/United States ; 1R43GM139439-01/GM/NIGMS NIH HHS/United States ; }, mesh = {Apoptosis/genetics ; CRISPR-Cas Systems/genetics ; Caspases/metabolism ; Cell Line, Tumor ; Humans ; *Melanoma/drug therapy/genetics/pathology ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; *Proto-Oncogene Proteins c-akt/metabolism ; Proto-Oncogene Proteins c-bcl-2/metabolism ; RNA, Small Interfering/pharmacology ; Skin Neoplasms ; Stem Cells/metabolism ; bcl-2-Associated X Protein/metabolism ; }, abstract = {Malignant melanoma is a lethal skin cancer containing melanoma-initiating cells (MIC) implicated in tumorigenesis, invasion, and drug resistance, and is characterized by the elevated expression of stem cell markers, including CD133. The siRNA knockdown of CD133 enhances apoptosis induced by the MEK inhibitor trametinib in melanoma cells. This study investigates the underlying mechanisms of CD133's anti-apoptotic activity in patient-derived BAKP and POT cells, harboring difficult-to-treat NRASQ61K and NRASQ61R drivers, after CRISPR-Cas9 CD133 knockout or Dox-inducible expression of CD133. MACS-sorted CD133(+) BAKP cells were conditionally reprogrammed to derive BAKR cells with sustained CD133 expression and MIC features. Compared to BAKP, CD133(+) BAKR exhibit increased cell survival and reduced apoptosis in response to trametinib or the chemotherapeutic dacarbazine (DTIC). CRISPR-Cas9-mediated CD133 knockout in BAKR cells (BAKR-KO) re-sensitized cells to trametinib. CD133 knockout in BAKP and POT cells increased trametinib-induced apoptosis by reducing anti-apoptotic BCL-xL, p-AKT, and p-BAD and increasing pro-apoptotic BAX. Conversely, Dox-induced CD133 expression diminished apoptosis in both trametinib-treated cell lines, coincident with elevated p-AKT, p-BAD, BCL-2, and BCL-xL and decreased activation of BAX and caspases-3 and -9. AKT1/2 siRNA knockdown or inhibition of BCL-2 family members with navitoclax (ABT-263) in BAKP-KO cells further enhanced caspase-mediated apoptotic PARP cleavage. CD133 may therefore activate a survival pathway where (1) increased AKT phosphorylation and activation induces (2) BAD phosphorylation and inactivation, (3) decreases BAX activation, and (4) reduces caspases-3 and -9 activity and caspase-mediated PARP cleavage, leading to apoptosis suppression and drug resistance in melanoma. Targeting nodes of the CD133, AKT, or BCL-2 survival pathways with trametinib highlights the potential for combination therapies for NRAS-mutant melanoma stem cells for the development of more effective treatments for patients with high-risk melanoma.}, }
@article {pmid35216418, year = {2022}, author = {Khan, ZA and Kumar, R and Dasgupta, I}, title = {CRISPR/Cas-Mediated Resistance against Viruses in Plants.}, journal = {International journal of molecular sciences}, volume = {23}, number = {4}, pages = {}, pmid = {35216418}, issn = {1422-0067}, support = {SB/S2/JCB-057/2016//J. C. Bose Fellowship, Science and Engineering Research Board, Government of India/ ; }, mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/methods ; Genome, Plant/*genetics ; Genome, Viral/*genetics ; Plant Viruses/*genetics ; Plants, Genetically Modified/genetics/virology ; }, abstract = {CRISPR/Cas9 provides a robust and widely adaptable system with enormous potential for genome editing directed towards generating useful products. It has been used extensively to generate resistance against viruses infecting plants with more effective and prolonged efficiency as compared with previous antiviral approaches, thus holding promise to alleviate crop losses. In this review, we have discussed the reports of CRISPR/Cas-based virus resistance strategies against plant viruses. These strategies include approaches targeting single or multiple genes (or non-coding region) in the viral genome and targeting host factors essential for virus propagation. In addition, the utilization of base editing has been discussed to generate transgene-free plants resistant to viruses. This review also compares the efficiencies of these approaches. Finally, we discuss combinatorial approaches, including multiplexing, to increase editing efficiency and bypass the generation of escape mutants.}, }
@article {pmid35216392, year = {2022}, author = {Tay Fernandez, CG and Nestor, BJ and Danilevicz, MF and Marsh, JI and Petereit, J and Bayer, PE and Batley, J and Edwards, D}, title = {Expanding Gene-Editing Potential in Crop Improvement with Pangenomes.}, journal = {International journal of molecular sciences}, volume = {23}, number = {4}, pages = {}, pmid = {35216392}, issn = {1422-0067}, support = {DP210100296//Australian Research Council/ ; DP200100762//Australian Research Council/ ; DE210100398//Australian Research Council/ ; 9177539//Grains Research and Development Corporation/ ; 9177591//Grains Research and Development Corporation/ ; }, mesh = {CRISPR-Cas Systems/*genetics ; Crops, Agricultural/*genetics ; Gene Editing/methods ; Genome, Plant/*genetics ; Phenotype ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; }, abstract = {Pangenomes aim to represent the complete repertoire of the genome diversity present within a species or cohort of species, capturing the genomic structural variance between individuals. This genomic information coupled with phenotypic data can be applied to identify genes and alleles involved with abiotic stress tolerance, disease resistance, and other desirable traits. The characterisation of novel structural variants from pangenomes can support genome editing approaches such as Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR associated protein Cas (CRISPR-Cas), providing functional information on gene sequences and new target sites in variant-specific genes with increased efficiency. This review discusses the application of pangenomes in genome editing and crop improvement, focusing on the potential of pangenomes to accurately identify target genes for CRISPR-Cas editing of plant genomes while avoiding adverse off-target effects. We consider the limitations of applying CRISPR-Cas editing with pangenome references and potential solutions to overcome these limitations.}, }
@article {pmid35216252, year = {2022}, author = {Navarro-Serna, S and Piñeiro-Silva, C and Luongo, C and Parrington, J and Romar, R and Gadea, J}, title = {Effect of Aphidicolin, a Reversible Inhibitor of Eukaryotic Nuclear DNA Replication, on the Production of Genetically Modified Porcine Embryos by CRISPR/Cas9.}, journal = {International journal of molecular sciences}, volume = {23}, number = {4}, pages = {}, pmid = {35216252}, issn = {1422-0067}, support = {DTS19/00061//Proyectos de Desarrollo Tecnológico AES 2019/ ; AGL 2015-66341-R//MINECO-FEDER/ ; 20040/GERM/16//Fundación Seneca/ ; 21105/PDC/19//Fundación Seneca/ ; 21666/PDC/21//Fundación Seneca/ ; FPU16/04480//Spanish Ministry of Education, Culture and Sport/ ; }, mesh = {Animals ; Animals, Genetically Modified ; Aphidicolin/*pharmacology ; CRISPR-Cas Systems/*drug effects ; Cell Nucleus/*drug effects ; DNA Replication/*drug effects ; Embryo, Mammalian/*drug effects ; Embryonic Development/drug effects ; Eukaryota/*drug effects ; Gene Editing/methods ; Mosaicism/drug effects ; Swine ; Zygote/drug effects ; }, abstract = {Mosaicism is the most important limitation for one-step gene editing in embryos by CRISPR/Cas9 because cuts and repairs sometimes take place after the first DNA replication of the zygote. To try to minimize the risk of mosaicism, in this study a reversible DNA replication inhibitor was used after the release of CRISPR/Cas9 in the cell. There is no previous information on the use of aphidicolin in porcine embryos, so the reversible inhibition of DNA replication and the effect on embryo development of different concentrations of this drug was first evaluated. The effect of incubation with aphidicolin was tested with CRISPR/Cas9 at different concentrations and different delivery methodologies. As a result, the reversible inhibition of DNA replication was observed, and it was concentration dependent. An optimal concentration of 0.5 μM was established and used for subsequent experiments. Following the use of this drug with CRISPR/Cas9, a halving of mosaicism was observed together with a detrimental effect on embryo development. In conclusion, the use of reversible inhibition of DNA replication offers a way to reduce mosaicism. Nevertheless, due to the reduction in embryo development, it would be necessary to reach a balance for its use to be feasible.}, }
@article {pmid35216118, year = {2022}, author = {Rahman, H and Fukushima, C and Kaya, H and Yaeno, T and Kobayashi, K}, title = {Knockout of Tobacco Homologs of Arabidopsis Multi-Antibiotic Resistance 1 Gene Confers a Limited Resistance to Aminoglycoside Antibiotics.}, journal = {International journal of molecular sciences}, volume = {23}, number = {4}, pages = {}, pmid = {35216118}, issn = {1422-0067}, support = {19K06055, 19K21150, 21K06233//Japan Society for the Promotion of Science/ ; Research Unit for Citromics//Ehime University/ ; }, mesh = {Agrobacterium/drug effects/genetics ; Aminoglycosides/*pharmacology ; Anti-Bacterial Agents/*pharmacology ; Arabidopsis/*genetics/microbiology ; CRISPR-Cas Systems/genetics ; Drug Resistance, Microbial/*genetics ; Gene Editing/methods ; Genome, Plant/genetics ; Mutation/genetics ; Plant Leaves/genetics/microbiology ; Plants, Genetically Modified/genetics ; RNA, Guide/genetics ; Tobacco/*genetics/microbiology ; }, abstract = {To explore a possible recessive selective marker for future DNA-free genome editing by direct delivery of a CRISPR/Cas9-single guide RNA (sgRNA) ribonucleoprotein complex, we knocked out homologs of the ArabidopsisMulti-Antibiotic Resistance 1 (MAR1)/RTS3 gene, mutations of which confer aminoglycoside resistance, in tobacco plants by an efficient Agrobacterium-mediated gene transfer. A Cas9 gene was introduced into Nicotiana tabacum and Nicotiana sylvestris together with an sgRNA gene for one of three different target sequences designed to perfectly match sequences in both S- and T-genome copies of N. tabacumMAR1 homologs (NtMAR1hs). All three sgRNAs directed the introduction of InDels into NtMAR1hs, as demonstrated by CAPS and amplicon sequencing analyses, albeit with varying efficiency. Leaves of regenerated transformant shoots were evaluated for aminoglycoside resistance on shoot-induction media containing different aminoglycoside antibiotics. All transformants tested were as sensitive to those antibiotics as non-transformed control plants, regardless of the mutation rates in NtMAR1hs. The NtMAR1hs-knockout seedlings of the T1 generation showed limited aminoglycoside resistance but failed to form shoots when cultured on shoot-induction media containing kanamycin. The results suggest that, like Arabidopsis MAR1, NtMAR1hs have a role in plants' sensitivity to aminoglycoside antibiotics, and that tobacco has some additional functional homologs.}, }
@article {pmid35216030, year = {2022}, author = {Prokhorova, D and Zhukova Eschenko, N and Lemza, A and Sergeeva, M and Amirkhanov, R and Stepanov, G}, title = {Application of the CRISPR/Cas9 System to Study Regulation Pathways of the Cellular Immune Response to Influenza Virus.}, journal = {Viruses}, volume = {14}, number = {2}, pages = {}, pmid = {35216030}, issn = {1999-4915}, mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line ; Chickens ; Gene Editing/*methods ; Humans ; Immunity, Cellular ; Influenza A virus/genetics/*physiology ; Virus Internalization ; Virus Replication ; }, abstract = {Influenza A virus (IAV) causes a respiratory infection that affects millions of people of different age groups and can lead to acute respiratory distress syndrome. Currently, host genes, receptors, and other cellular components critical for IAV replication are actively studied. One of the most convenient and accessible genome-editing tools to facilitate these studies is the CRISPR/Cas9 system. This tool allows for regulating the expression of both viral and host cell genes to enhance or impair viral entry and replication. This review considers the effect of the genome editing system on specific target genes in cells (human and chicken) in terms of subsequent changes in the influenza virus life cycle and the efficiency of virus particle production.}, }
@article {pmid35216029, year = {2022}, author = {Li, W and Zhang, Y and Moffat, K and Nair, V and Yao, Y}, title = {V5 and GFP Tagging of Viral Gene pp38 of Marek's Disease Vaccine Strain CVI988 Using CRISPR/Cas9 Editing.}, journal = {Viruses}, volume = {14}, number = {2}, pages = {}, pmid = {35216029}, issn = {1999-4915}, support = {BBS/E/I/00007039/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; }, mesh = {Animals ; CRISPR-Cas Systems ; Chickens/virology ; Gene Editing/*methods ; Genome, Viral ; Green Fluorescent Proteins/*genetics ; Mardivirus/*genetics ; Marek Disease/prevention &amp; control ; Marek Disease Vaccines/*genetics ; Phosphoproteins/genetics ; Poultry Diseases/prevention &amp; control ; Viral Envelope Proteins/chemistry/*genetics ; Virus Replication ; }, abstract = {Marek's disease virus (MDV) is a member of alphaherpesviruses associated with Marek's disease, a highly contagious neoplastic disease in chickens. The availability of the complete sequence of the viral genome allowed for the identification of major genes associated with pathogenicity using different techniques, such as bacterial artificial chromosome (BAC) mutagenesis and the recent powerful clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based editing system. Thus far, most studies on MDV genome editing using the CRISPR/Cas9 system have focused on gene deletion. However, analysis of the expression and interactions of the viral proteins during virus replication in infected cells and tumor cells is also important for studying its role in MDV pathogenesis. The unavailability of antibodies against most of the MDV proteins has hindered the progress in such studies. This prompted us to develop pipelines to tag MDV genes as an alternative method for this purpose. Here we describe the application of CRISPR/Cas9 gene-editing approaches to tag the phosphoprotein 38 (pp38) gene of the MDV vaccine strain CVI988 with both V5 and green fluorescent protein (GFP). This rapid and efficient viral-gene-tagging technique can overcome the shortage of specific antibodies and speed up the MDV gene function studies significantly, leading to a better understanding of the molecular mechanisms of MDV pathogenesis.}, }
@article {pmid35216010, year = {2022}, author = {Yuan, H and Yang, L and Zhang, Y and Xiao, W and Wang, Z and Tang, X and Ouyang, H and Pang, D}, title = {Current Status of Genetically Modified Pigs That Are Resistant to Virus Infection.}, journal = {Viruses}, volume = {14}, number = {2}, pages = {}, pmid = {35216010}, issn = {1999-4915}, mesh = {African Swine Fever Virus/*genetics ; Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/genetics ; Classical Swine Fever Virus/*genetics ; Gene Editing/methods ; Herpesvirus 1, Suid/*genetics ; Porcine epidemic diarrhea virus/*genetics ; Porcine respiratory and reproductive syndrome virus/*genetics ; Swine/virology ; Transmissible gastroenteritis virus/*genetics ; Virus Diseases/prevention &amp; control ; }, abstract = {Pigs play an important role in agriculture and biomedicine. The globally developing swine industry must address the challenges presented by swine-origin viruses, including ASFV (African swine fever virus), PRRSV (porcine reproductive and respiratory syndrome virus), PEDV (porcine epidemic diarrhea virus), PRV (pseudorabies virus), CSFV (classical swine fever virus), TGEV (transmissible gastroenteritis virus), et al. Despite sustained efforts by many government authorities, these viruses are still widespread. Currently, gene-editing technology has been successfully used to generate antiviral pigs, which offers the possibility for increasing animal disease tolerance and improving animal economic traits in the future. Here, we summarized the current advance in knowledge regarding the host factors in virus infection and the current status of genetically modified pigs that are resistant to virus infection in the world. There has not been any report on PEDV-resistant pigs, ASFV-resistant pigs, and PRV-resistant pigs owing to the poor understanding of the key host factors in virus infection. Furthermore, we summarized the remaining problems in producing virus-resistant pigs, and proposed several potential methods to solve them. Using genome-wide CRISPR/Cas9 library screening to explore the key host receptors in virus infection may be a feasible method. At the same time, exploring the key amino acids of host factors in virus infection with library screening based on ABEs and CBEs (Bes) may provide creative insight into producing antiviral pigs in the future.}, }
@article {pmid35215977, year = {2022}, author = {Hussein, M and Andrade Dos Ramos, Z and Berkhout, B and Herrera-Carrillo, E}, title = {In Silico Prediction and Selection of Target Sequences in the SARS-CoV-2 RNA Genome for an Antiviral Attack.}, journal = {Viruses}, volume = {14}, number = {2}, pages = {}, pmid = {35215977}, issn = {1999-4915}, support = {Aspasia-NWO 015.015.040//Nederlandse Organisatie voor Wetenschappelijk Onderzoek/ ; }, mesh = {CRISPR-Cas Systems ; *Computer Simulation ; *Genome, Viral ; Humans ; RNA, Viral/*genetics ; SARS-CoV-2/*genetics ; Spike Glycoprotein, Coronavirus/genetics ; }, abstract = {The SARS-CoV-2 pandemic has urged the development of protective vaccines and the search for specific antiviral drugs. The modern molecular biology tools provides alternative methods, such as CRISPR-Cas and RNA interference, that can be adapted as antiviral approaches, and contribute to this search. The unique CRISPR-Cas13d system, with the small crRNA guide molecule, mediates a sequence-specific attack on RNA, and can be developed as an anti-coronavirus strategy. We analyzed the SARS-CoV-2 genome to localize the hypothetically best crRNA-annealing sites of 23 nucleotides based on our extensive expertise with sequence-specific antiviral strategies. We considered target sites of which the sequence is well-conserved among SARS-CoV-2 isolates. As we should prepare for a potential future outbreak of related viruses, we screened for targets that are conserved between SARS-CoV-2 and SARS-CoV. To further broaden the search, we screened for targets that are conserved between SARS-CoV-2 and the more distantly related MERS-CoV, as well as the four other human coronaviruses (OC43, 229E, NL63, HKU1). Finally, we performed a search for pan-corona target sequences that are conserved among all these coronaviruses, including the new Omicron variant, that are able to replicate in humans. This survey may contribute to the design of effective, safe, and escape-proof antiviral strategies to prepare for future pandemics.}, }
@article {pmid35215971, year = {2022}, author = {Wu, BW and Yee, MB and Goldstein, RS and Kinchington, PR}, title = {Antiviral Targeting of Varicella Zoster Virus Replication and Neuronal Reactivation Using CRISPR/Cas9 Cleavage of the Duplicated Open Reading Frames 62/71.}, journal = {Viruses}, volume = {14}, number = {2}, pages = {}, pmid = {35215971}, issn = {1999-4915}, support = {R01 AI122640/AI/NIAID NIH HHS/United States ; T32 AI049820/AI/NIAID NIH HHS/United States ; }, mesh = {Antiviral Agents/pharmacology ; *CRISPR-Cas Systems ; Cell Line ; Dependovirus/*genetics ; Drug Discovery ; Herpesvirus 3, Human/drug effects/*physiology ; Human Embryonic Stem Cells ; Humans ; Immediate-Early Proteins ; Neurons/*virology ; Open Reading Frames/*genetics ; Trans-Activators ; Viral Envelope Proteins ; Virus Latency ; Virus Replication ; }, abstract = {Varicella Zoster Virus (VZV) causes Herpes Zoster (HZ), a common debilitating and complicated disease affecting up to a third of unvaccinated populations. Novel antiviral treatments for VZV reactivation and HZ are still in need. Here, we evaluated the potential of targeting the replicating and reactivating VZV genome using Clustered Regularly Interspaced Short Palindromic Repeat-Cas9 nucleases (CRISPR/Cas9) delivered by adeno-associated virus (AAV) vectors. After AAV serotype and guide RNA (gRNA) optimization, we report that a single treatment with AAV2-expressing Staphylococcus aureus CRISPR/Cas9 (saCas9) with gRNA to the duplicated and essential VZV genes ORF62/71 (AAV2-62gRsaCas9) greatly reduced VZV progeny yield and cell-to-cell spread in representative epithelial cells and in lytically infected human embryonic stem cell (hESC)-derived neurons. In contrast, AAV2-62gRsaCas9 did not reduce the replication of a recombinant virus mutated in the ORF62 targeted sequence, establishing that antiviral effects were a consequence of VZV-genome targeting. Delivery to latently infected and reactivation-induced neuron cultures also greatly reduced infectious-virus production. These results demonstrate the potential of AAV-delivered genome editors to limit VZV productive replication in epithelial cells, infected human neurons, and upon reactivation. The approach could be developed into a strategy for the treatment of VZV disease and virus spread in HZ.}, }
@article {pmid35215968, year = {2022}, author = {Lu, H and Xie, Q and Zhang, W and Zhang, J and Wang, W and Lian, M and Zhao, Z and Ren, D and Xie, S and Lin, Y and Li, T and Mu, Y and Wan, Z and Shao, H and Qin, A and Ye, J}, title = {A Novel Recombinant FAdV-4 Virus with Fiber of FAdV-8b Provides Efficient Protection against Both FAdV-4 and FAdV-8b.}, journal = {Viruses}, volume = {14}, number = {2}, pages = {}, pmid = {35215968}, issn = {1999-4915}, mesh = {Adenoviridae Infections/*prevention &amp; control/veterinary/virology ; Animals ; Antibodies, Neutralizing/blood ; Aviadenovirus/*genetics ; CRISPR-Cas Systems ; *Chickens ; Gene Editing ; Poultry Diseases/*prevention &amp; control/virology ; Serogroup ; Vaccines, Synthetic/administration &amp; dosage ; Viral Vaccines/*administration &amp; dosage ; }, abstract = {Since 2015, the outbreaks of hydropericardium-hepatitis syndrome (HHS) and inclusion body hepatitis (IBH) caused by the highly pathogenic serotype 4 fowl adenovirus (FAdV-4) and serotype 8 fowl adenovirus (FAdV-8), respectively, have caused huge economic losses to the poultry industry. Although several vaccines have been developed to control HHS or IBH, a recombinant genetic engineering vaccine against both FAdV-4 and FAdV-8 has not been reported. In this study, recombinant FAdV-4 expressing the fiber of FAdV-8b, designated as FA4-F8b, expressing fiber of FAdV-8b was generated by the CRISPR-Cas9 and homologous recombinant techniques. Infection studies in vitro and in vivo revealed that the FA4-F8b replicated efficiently in LMH cells and was also highly pathogenic to 2-week-old SPF chickens. Moreover, the inoculation of inactivated the FA4-F8b in chickens could not only induce highly neutralizing antibodies, but also provide efficient protection against both FAdV-4 and FAdV-8b. All these demonstrate that the inactivated recombinant FA4-F8b generated here can act as a vaccine candidate to control HHS and IBH, and FAdV-4 can be an efficient vaccine vector to deliver foreign antigens.}, }
@article {pmid35215773, year = {2022}, author = {Wei, N and Zheng, B and Niu, J and Chen, T and Ye, J and Si, Y and Cao, S}, title = {Rapid Detection of Genotype II African Swine Fever Virus Using CRISPR Cas13a-Based Lateral Flow Strip.}, journal = {Viruses}, volume = {14}, number = {2}, pages = {}, pmid = {35215773}, issn = {1999-4915}, mesh = {African Swine Fever/*diagnosis/virology ; African Swine Fever Virus/genetics/*isolation &amp; purification ; Animals ; *CRISPR-Cas Systems ; Genotype ; Reagent Strips ; Recombinases/genetics/metabolism ; Sensitivity and Specificity ; Sus scrofa ; Swine ; Time Factors ; }, abstract = {The African swine fever virus (ASFV) is a dsDNA virus that can cause serious, highly infectious, and fatal diseases in wild boars and domestic pigs. The ASFV has brought enormous economic loss to many countries, and no effective vaccine or treatment for the ASFV is currently available. Therefore, the on-site rapid and accurate detection of the ASFV is key to the timely implementation of control. The RNA-guided, RNA-targeting CRISPR effector CRISPR-associated 13 (Cas13a; previously known as C2c2) exhibits a "collateral effect" of promiscuous RNase activity upon the target recognition. The collateral cleavage activity of LwCas13a is activated to degrade the non-targeted RNA, when the crRNA of LwCas13a binds to the target RNA. In this study, we developed a rapid and sensitive ASFV detection method based on the collateral cleavage activity of LwCas13a, which combines recombinase-aided amplification (RAA) and a lateral flow strip (named CRISPR/Cas13a-LFD). The method was an isothermal detection at 37 °C, and the detection can be used for visual readout. The detection limit of the CRISPR/Cas13a-LFD was 101 copies/µL of p72 gene per reaction, and the detection process can be completed within an hour. The assay showed no cross-reactivity to eight other swine viruses, including classical swine fever virus (CSFV), and has a 100% coincidence rate with real-time PCR detection of the ASFV in 83 clinical samples. Overall, this method is sensitive, specific, and practicable onsite for the ASFV detection, showing a great application potential for monitoring the ASFV in the field.}, }
@article {pmid35213016, year = {2022}, author = {López-Márquez, A and Martínez-Pizarro, A and Pérez, B and Richard, E and Desviat, LR}, title = {Modeling Splicing Variants Amenable to Antisense Therapy by Use of CRISPR-Cas9-Based Gene Editing in HepG2 Cells.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2434}, number = {}, pages = {167-184}, pmid = {35213016}, issn = {1940-6029}, mesh = {*CRISPR-Cas Systems/genetics ; Exons/genetics ; *Gene Editing/methods ; Hep G2 Cells ; Humans ; RNA Splicing ; }, abstract = {The field of splice modulating RNA therapy has gained new momentum with FDA approved antisense-based drugs for several rare diseases. In vitro splicing assays with minigenes or patient-derived cells are commonly employed for initial preclinical testing of antisense oligonucleotides aiming to modulate splicing. However, minigenes do not include the full genomic context of the exons under study and patients' samples are not always available, especially if the gene is expressed solely in certain tissues (e.g. liver or brain). This is the case for specific inherited metabolic diseases such as phenylketonuria (PKU) caused by mutations in the liver-expressed PAH gene.Herein we describe the generation of mutation-specific hepatic cellular models of PKU using CRISPR/Cas9 system, which is a versatile and easy-to-use gene editing tool. We describe in detail the selection of the appropriate cell line, guidelines for design of RNA guides and donor templates, transfection procedures and growth and selection of single-cell colonies with the desired variant , which should result in the accurate recapitulation of the splicing defect.}, }
@article {pmid35209078, year = {2022}, author = {Skripova, V and Vlasenkova, R and Zhou, Y and Astsaturov, I and Kiyamova, R}, title = {Identification of New Regulators of Pancreatic Cancer Cell Sensitivity to Oxaliplatin and Cisplatin.}, journal = {Molecules (Basel, Switzerland)}, volume = {27}, number = {4}, pages = {}, pmid = {35209078}, issn = {1420-3049}, support = {//This paper has been supported by the Kazan Federal University Strategic Academic Leadership Program/ ; }, mesh = {Antineoplastic Agents/*pharmacology ; Biomarkers, Tumor ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cisplatin/*pharmacology ; Computational Biology/methods ; Drug Resistance, Neoplasm/*drug effects/genetics ; Drug Screening Assays, Antitumor ; Gene Expression Profiling ; Gene Knockdown Techniques ; Gene Ontology ; Gene Regulatory Networks ; Humans ; Oxaliplatin/*pharmacology ; Pancreatic Neoplasms ; Synthetic Lethal Mutations ; }, abstract = {The chemoresistance of tumor cells is one of the most urgent challenges in modern oncology and in pancreatic cancer, in which this problem is the most prominent. Therefore, the identification of new chemosensitizing co-targets may be a path toward increasing chemotherapy efficacy. In this work, we performed high-performance in vitro knockout CRISPR/Cas9 screening to find potential regulators of the sensitivity of pancreatic cancer. For this purpose, MIA PaCa-2 cells transduced with two sgRNA libraries ("cell cycle/nuclear proteins genes" and "genome-wide") were screened by oxaliplatin and cisplatin. In total, 173 candidate genes were identified as potential regulators of pancreatic cancer cell sensitivity to oxaliplatin and/or cisplatin; among these, 25 genes have previously been reported, while 148 genes were identified for the first time as potential platinum drug sensitivity regulators. We found seven candidate genes involved in pancreatic cancer cell sensitivity to both cisplatin and oxaliplatin. Gene ontology enrichment analysis reveals the enrichment of single-stranded DNA binding, damaged DNA binding pathways, and four associated with NADH dehydrogenase activity. Further investigation and validation of the obtained results by in vitro, in vivo, and bioinformatics approaches, as well as literature analysis, will help to identify novel pancreatic cancer platinum sensitivity regulators.}, }
@article {pmid35207459, year = {2022}, author = {Gutierrez, K and Glanzner, WG and de Macedo, MP and Rissi, VB and Dicks, N and Bohrer, RC and Baldassarre, H and Agellon, LB and Bordignon, V}, title = {Cell Cycle Stage and DNA Repair Pathway Influence CRISPR/Cas9 Gene Editing Efficiency in Porcine Embryos.}, journal = {Life (Basel, Switzerland)}, volume = {12}, number = {2}, pages = {}, pmid = {35207459}, issn = {2075-1729}, support = {RGPIN/04910-2016//Natural Sciences and Engineering Research Council/ ; RGPIN-2015-04390//Natural Sciences and Engineering Research Council/ ; 010000/2013-05//Coordenação de Aperfeicoamento de Pessoal de Nível Superior/ ; }, abstract = {CRISPR/Cas9 technology is a powerful tool used for genome manipulation in different cell types and species. However, as with all new technologies, it still requires improvements. Different factors can affect CRISPR/Cas efficiency in zygotes, which influence the total cost and complexity for creating large-animal models for research. This study evaluated the importance of zygote cell cycle stage between early-injection (within 6 h post activation/fertilization) versus late-injection (14-16 h post activation/fertilization) when the CRISPR/Cas9 components were injected and the inhibition of the homologous recombination (HR) pathway of DNA repair on gene editing, embryo survival and development on embryos produced by fertilization, sperm injection, somatic cell nuclear transfer, and parthenogenetic activation technologies. Injections at the late cell cycle stage decreased embryo survival (measured as the proportion of unlysed embryos) and blastocyst formation (68.2%; 19.3%) compared to early-stage injection (86.3%; 28.8%). However, gene editing was higher in blastocysts from late-(73.8%) vs. early-(63.8%) injected zygotes. Inhibition of the HR repair pathway increased gene editing efficiency by 15.6% in blastocysts from early-injected zygotes without compromising embryo development. Our finding shows that injection at the early cell cycle stage along with HR inhibition improves both zygote viability and gene editing rate in pig blastocysts.}, }
@article {pmid35203804, year = {2022}, author = {Murugaiyan, J and Kumar, PA and Rao, GS and Iskandar, K and Hawser, S and Hays, JP and Mohsen, Y and Adukkadukkam, S and Awuah, WA and Jose, RAM and Sylvia, N and Nansubuga, EP and Tilocca, B and Roncada, P and Roson-Calero, N and Moreno-Morales, J and Amin, R and Kumar, BK and Kumar, A and Toufik, AR and Zaw, TN and Akinwotu, OO and Satyaseela, MP and van Dongen, MBM}, title = {Progress in Alternative Strategies to Combat Antimicrobial Resistance: Focus on Antibiotics.}, journal = {Antibiotics (Basel, Switzerland)}, volume = {11}, number = {2}, pages = {}, pmid = {35203804}, issn = {2079-6382}, abstract = {Antibiotic resistance, and, in a broader perspective, antimicrobial resistance (AMR), continues to evolve and spread beyond all boundaries. As a result, infectious diseases have become more challenging or even impossible to treat, leading to an increase in morbidity and mortality. Despite the failure of conventional, traditional antimicrobial therapy, in the past two decades, no novel class of antibiotics has been introduced. Consequently, several novel alternative strategies to combat these (multi-) drug-resistant infectious microorganisms have been identified. The purpose of this review is to gather and consider the strategies that are being applied or proposed as potential alternatives to traditional antibiotics. These strategies include combination therapy, techniques that target the enzymes or proteins responsible for antimicrobial resistance, resistant bacteria, drug delivery systems, physicochemical methods, and unconventional techniques, including the CRISPR-Cas system. These alternative strategies may have the potential to change the treatment of multi-drug-resistant pathogens in human clinical settings.}, }
@article {pmid35203404, year = {2022}, author = {Haase-Kohn, C and Laube, M and Donat, CK and Belter, B and Pietzsch, J}, title = {CRISPR/Cas9 Mediated Knockout of Cyclooxygenase-2 Gene Inhibits Invasiveness in A2058 Melanoma Cells.}, journal = {Cells}, volume = {11}, number = {4}, pages = {}, pmid = {35203404}, issn = {2073-4409}, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Cyclooxygenase 2/genetics/metabolism ; Humans ; *Melanoma/pathology ; Mice ; Mice, Knockout ; Tumor Microenvironment ; }, abstract = {The inducible isoenzyme cyclooxygenase-2 (COX-2) is an important hub in cellular signaling, which contributes to tumor progression by modulating and enhancing a pro-inflammatory tumor microenvironment, tumor growth, apoptosis resistance, angiogenesis and metastasis. In order to understand the role of COX-2 expression in melanoma, we investigated the functional knockout effect of COX-2 in A2058 human melanoma cells. COX-2 knockout was validated by Western blot and flow cytometry analysis. When comparing COX-2 knockout cells to controls, we observed significantly reduced invasion, colony and spheroid formation potential in cell monolayers and three-dimensional models in vitro, and significantly reduced tumor development in xenograft mouse models in vivo. Moreover, COX-2 knockout alters the metabolic activity of cells under normoxia and experimental hypoxia as demonstrated by using the radiotracers [18F]FDG and [18F]FMISO. Finally, a pilot protein array analysis in COX-2 knockout cells verified significantly altered downstream signaling pathways that can be linked to cellular and molecular mechanisms of cancer metastasis closely related to the enzyme. Given the complexity of the signaling pathways and the multifaceted role of COX-2, targeted suppression of COX-2 in melanoma cells, in combination with modulation of related signaling pathways, appears to be a promising therapeutic approach.}, }
@article {pmid35202423, year = {2022}, author = {Addo-Gyan, D and Matsushita, H and Sora, E and Nishi, T and Yuda, M and Shinzawa, N and Iwanaga, S}, title = {Chromosome splitting of Plasmodium berghei using the CRISPR/Cas9 system.}, journal = {PloS one}, volume = {17}, number = {2}, pages = {e0260176}, pmid = {35202423}, issn = {1932-6203}, mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Centromere/genetics ; Chromosomes/*genetics ; Gene Expression Regulation/genetics ; Malaria/*genetics/parasitology ; Plasmodium berghei/*genetics/pathogenicity ; Plasmodium falciparum/genetics/pathogenicity ; Rodentia/parasitology ; Telomere/genetics ; }, abstract = {Spatial arrangement of chromosomes is responsible for gene expression in Plasmodium parasites. However, methods for rearranging chromosomes have not been established, which makes it difficult to investigate its role in detail. Here, we report a method for splitting chromosome in rodent malaria parasite by CRISPR/Cas9 system using fragments in which a telomere and a centromere were incorporated. The resultant split chromosomes segregated accurately into daughter parasites by the centromere. In addition, elongation of de novo telomeres were observed, indicating its proper function. Furthermore, chromosome splitting had no effect on development of parasites. Splitting of the chromosome is expected to alter its spatial arrangement, and our method will thus be useful for investigating its biological role related with gene expression.}, }
@article {pmid35201886, year = {2022}, author = {Li, SB and Damonte, VM and Chen, C and Wang, GX and Kebschull, JM and Yamaguchi, H and Bian, WJ and Purmann, C and Pattni, R and Urban, AE and Mourrain, P and Kauer, JA and Scherrer, G and de Lecea, L}, title = {Hyperexcitable arousal circuits drive sleep instability during aging.}, journal = {Science (New York, N.Y.)}, volume = {375}, number = {6583}, pages = {eabh3021}, pmid = {35201886}, issn = {1095-9203}, support = {R01 NS106301/NS/NINDS NIH HHS/United States ; P30 EY026877/EY/NEI NIH HHS/United States ; K01 AG061230/AG/NIA NIH HHS/United States ; R01 MH116470/MH/NIMH NIH HHS/United States ; R01 NS104950/NS/NINDS NIH HHS/United States ; P50 HG007735/HG/NHGRI NIH HHS/United States ; R01 DA011289/DA/NIDA NIH HHS/United States ; R01 AG047671/AG/NIA NIH HHS/United States ; R01 MH128140/MH/NIMH NIH HHS/United States ; P50 AG047366/AG/NIA NIH HHS/United States ; }, mesh = {*Aging ; Aminopyridines/pharmacology ; Animals ; CRISPR-Cas Systems ; Electroencephalography ; Electromyography ; Female ; Hypothalamic Area, Lateral/physiopathology ; KCNQ2 Potassium Channel/genetics/metabolism ; KCNQ3 Potassium Channel/genetics/metabolism ; Male ; Mice ; Narcolepsy/genetics/physiopathology ; Nerve Tissue Proteins/genetics/metabolism ; Neural Pathways ; Neurons/*physiology ; Optogenetics ; Orexins/*physiology ; Patch-Clamp Techniques ; RNA-Seq ; *Sleep ; Sleep Deprivation/*physiopathology ; Sleep Quality ; *Wakefulness ; }, abstract = {Sleep quality declines with age; however, the underlying mechanisms remain elusive. We found that hyperexcitable hypocretin/orexin (Hcrt/OX) neurons drive sleep fragmentation during aging. In aged mice, Hcrt neurons exhibited more frequent neuronal activity epochs driving wake bouts, and optogenetic activation of Hcrt neurons elicited more prolonged wakefulness. Aged Hcrt neurons showed hyperexcitability with lower KCNQ2 expression and impaired M-current, mediated by KCNQ2/3 channels. Single-nucleus RNA-sequencing revealed adaptive changes to Hcrt neuron loss in the aging brain. Disruption of Kcnq2/3 genes in Hcrt neurons of young mice destabilized sleep, mimicking aging-associated sleep fragmentation, whereas the KCNQ-selective activator flupirtine hyperpolarized Hcrt neurons and rejuvenated sleep architecture in aged mice. Our findings demonstrate a mechanism underlying sleep instability during aging and a strategy to improve sleep continuity.}, }
@article {pmid35200314, year = {2022}, author = {Díaz-Galicia, E and Grünberg, R and Arold, ST}, title = {How to Find the Right RNA-Sensing CRISPR-Cas System for an In Vitro Application.}, journal = {Biosensors}, volume = {12}, number = {2}, pages = {}, pmid = {35200314}, issn = {2079-6374}, support = {REI/1/4204-01//King Abdullah University of Science and Technology/ ; }, mesh = {*CRISPR-Cas Systems ; *RNA ; }, abstract = {CRISPR-Cas systems have a great and still largely untapped potential for in vitro applications, in particular, for RNA biosensing. However, there is currently no systematic guide on selecting the most appropriate RNA-targeting CRISPR-Cas system for a given application among thousands of potential candidates. We provide an overview of the currently described Cas effector systems and review existing Cas-based RNA detection methods. We then propose a set of systematic selection criteria for selecting CRISPR-Cas candidates for new applications. Using this approach, we identify four candidates for in vitro RNA.}, }
@article {pmid35198566, year = {2021}, author = {Feng, S and Wang, Z and Li, A and Xie, X and Liu, J and Li, S and Li, Y and Wang, B and Hu, L and Yang, L and Guo, T}, title = {Strategies for High-Efficiency Mutation Using the CRISPR/Cas System.}, journal = {Frontiers in cell and developmental biology}, volume = {9}, number = {}, pages = {803252}, pmid = {35198566}, issn = {2296-634X}, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated systems have revolutionized traditional gene-editing tools and are a significant tool for ameliorating gene defects. Characterized by high target specificity, extraordinary efficiency, and cost-effectiveness, CRISPR/Cas systems have displayed tremendous potential for genetic manipulation in almost any organism and cell type. Despite their numerous advantages, however, CRISPR/Cas systems have some inherent limitations, such as off-target effects, unsatisfactory efficiency of delivery, and unwanted adverse effects, thereby resulting in a desire to explore approaches to address these issues. Strategies for improving the efficiency of CRISPR/Cas-induced mutations, such as reducing off-target effects, improving the design and modification of sgRNA, optimizing the editing time and the temperature, choice of delivery system, and enrichment of sgRNA, are comprehensively described in this review. Additionally, several newly emerging approaches, including the use of Cas variants, anti-CRISPR proteins, and mutant enrichment, are discussed in detail. Furthermore, the authors provide a deep analysis of the current challenges in the utilization of CRISPR/Cas systems and the future applications of CRISPR/Cas systems in various scenarios. This review not only serves as a reference for improving the maturity of CRISPR/Cas systems but also supplies practical guidance for expanding the applicability of this technology.}, }
@article {pmid35197604, year = {2022}, author = {Lin, SC and Haga, K and Zeng, XL and Estes, MK}, title = {Generation of CRISPR-Cas9-mediated genetic knockout human intestinal tissue-derived enteroid lines by lentivirus transduction and single-cell cloning.}, journal = {Nature protocols}, volume = {17}, number = {4}, pages = {1004-1027}, pmid = {35197604}, issn = {1750-2799}, support = {PO1AI057788//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; U19 AI144297/AI/NIAID NIH HHS/United States ; P42ES0327725//U.S. Department of Health &amp; Human Services | NIH | National Institute of Environmental Health Sciences (NIEHS)/ ; U19 AI116497/AI/NIAID NIH HHS/United States ; CA125123//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; P01 AI057788/AI/NIAID NIH HHS/United States ; RP160283//Cancer Prevention and Research Institute of Texas (Cancer Prevention Research Institute of Texas)/ ; P42 ES027725/ES/NIEHS NIH HHS/United States ; P30 ES030285/ES/NIEHS NIH HHS/United States ; P30 CA125123/CA/NCI NIH HHS/United States ; P30 DK056338/DK/NIDDK NIH HHS/United States ; U19AI144297//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; P30DK56338//U.S. Department of Health &amp; Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes &amp; Digestive &amp; Kidney Diseases)/ ; P30ES030285//U.S. Department of Health &amp; Human Services | NIH | National Institute of Environmental Health Sciences (NIEHS)/ ; HHSN2722017000381//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; HHSN272201700081C/AI/NIAID NIH HHS/United States ; U19AI116497//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; RP17005//Cancer Prevention and Research Institute of Texas (Cancer Prevention Research Institute of Texas)/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; Clone Cells ; Cloning, Molecular ; Gene Editing/methods ; Gene Knockout Techniques ; Humans ; *Lentivirus/genetics ; RNA, Guide/genetics ; }, abstract = {Human intestinal tissue-derived enteroids (HIEs; also called organoids) are a powerful ex vivo model for gastrointestinal research. Genetic modification of these nontransformed cultures allows new insights into gene function and biological processes involved in intestinal diseases as well as gastrointestinal and donor segment-specific function. Here we provide a detailed technical pipeline and protocol for using the CRISPR-Cas9 genome editing system to knock out a gene of interest specifically in HIEs by lentiviral transduction and single-cell cloning. This protocol differs from a previously published alternative using electroporation of human colonoids to deliver piggyback transposons or CRISPR-Cas9 constructs, as this protocol uses a modified, fused LentiCRISPRv2-small-guiding RNA to express Cas9 and small-guiding RNA in a lentivirus. The protocol also includes the steps of gene delivery and subsequent single-cell cloning of the knockout cells as well as verification of clones and sequence identification of the mutation sites to establish knockout clones. An overview flowchart, step-by-step guidelines and troubleshooting suggestions are provided to aid the researcher in obtaining the genetic knockout HIE line within 2-3 months. In this protocol, we further describe how to use HIEs as an ex vivo model to assess host restriction factors for viral replication (using human norovirus replication as an example) by knocking out host attachment factors or innate immunity genes. Other applications are discussed to broaden the utility of this system, for example, to generate knockin or conditional knockout HIE lines to investigate the function of essential genes in many biological processes including other types of organoids.}, }
@article {pmid35197298, year = {2022}, author = {Xu, B and Kucenas, S and Zong, H}, title = {zMADM (zebrafish mosaic analysis with double markers) for single-cell gene knockout and dual-lineage tracing.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {119}, number = {9}, pages = {}, pmid = {35197298}, issn = {1091-6490}, support = {P30 CA044579/CA/NCI NIH HHS/United States ; R21 OD026524/OD/NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems ; *Cell Lineage ; Gene Knockdown Techniques ; *Genetic Markers ; *Mosaicism ; Single-Cell Analysis/*methods ; Zebrafish/*genetics ; }, abstract = {As a vertebrate model organism, zebrafish has many unique advantages in developmental studies, regenerative biology, and disease modeling. However, tissue-specific gene knockout in zebrafish is challenging due to technical difficulties in making floxed alleles. Even when successful, tissue-level knockout can affect too many cells, making it difficult to distinguish cell autonomous from noncell autonomous gene function. Here, we present a genetic system termed zebrafish mosaic analysis with double markers (zMADM). Through Cre/loxP-mediated interchromosomal mitotic recombination of two reciprocally chimeric fluorescent genes, zMADM generates sporadic (<0.5%), GFP+ mutant cells along with RFP+ sibling wild-type cells, enabling phenotypic analysis at single-cell resolution. Using wild-type zMADM, we traced two sibling cells (GFP+ and RFP+) in real time during a dynamic developmental process. Using nf1 mutant zMADM, we demonstrated an overproliferation phenotype of nf1 mutant cells in comparison to wild-type sibling cells in the same zebrafish. The readiness of zMADM to produce sporadic mutant cells without the need to generate floxed alleles should fundamentally improve the throughput of genetic analysis in zebrafish; the lineage-tracing capability combined with phenotypic analysis at the single-cell level should lead to deep insights into developmental and disease mechanisms. Therefore, we are confident that zMADM will enable groundbreaking discoveries once broadly distributed in the field.}, }
@article {pmid35196804, year = {2022}, author = {Shahi, I and Llaneras, CN and Perelman, SS and Torres, VJ and Ratner, AJ}, title = {Genome-Wide CRISPR-Cas9 Screen Does Not Identify Host Factors Modulating Streptococcus agalactiae β-Hemolysin/Cytolysin-Induced Cell Death.}, journal = {Microbiology spectrum}, volume = {10}, number = {1}, pages = {e0218621}, pmid = {35196804}, issn = {2165-0497}, support = {P30 CA016087/CA/NCI NIH HHS/United States ; R01 AI155476/AI/NIAID NIH HHS/United States ; R01 AI099394/AI/NIAID NIH HHS/United States ; R01 AI105129/AI/NIAID NIH HHS/United States ; R01 AI143290/AI/NIAID NIH HHS/United States ; R01 AI121244/AI/NIAID NIH HHS/United States ; R21 AI147511/AI/NIAID NIH HHS/United States ; }, mesh = {CD59 Antigens/genetics/metabolism ; CRISPR-Cas Systems ; Cell Death ; Cell Line ; Genome, Bacterial ; Hemolysin Proteins/metabolism/*toxicity ; Host-Pathogen Interactions ; Humans ; Perforin/metabolism/*toxicity ; Streptococcal Infections/*genetics/metabolism/microbiology/*physiopathology ; Streptococcus agalactiae/genetics/*metabolism ; }, abstract = {Pore-forming toxins (PFTs) are commonly produced by pathogenic bacteria, and understanding them is key to the development of virulence-targeted therapies. Streptococcus agalactiae, or group B Streptococcus (GBS), produces several factors that enhance its pathogenicity, including the PFT β-hemolysin/cytolysin (βhc). Little is understood about the cellular factors involved in βhc pore formation. We conducted a whole-genome CRISPR-Cas9 forward genetic screen to identify host genes that might contribute to βhc pore formation and cell death. While the screen identified the established receptor, CD59, in control experiments using the toxin intermedilysin (ILY), no clear candidate genes were identified that were required for βhc-mediated lethality. Of the top targets from the screen, two genes involved in membrane remodeling and repair represented candidates that might modulate the kinetics of βhc-induced cell death. Upon attempted validation of the results using monoclonal cell lines with targeted disruption of these genes, no effect on βhc-mediated cell lysis was observed. The CRISPR-Cas9 screen results are consistent with the hypothesis that βhc does not require a single nonessential host factor to mediate target cell death. IMPORTANCE CRISPR-Cas9 forward genetic screens have been used to identify host cell targets required by bacterial toxins. They have been used successfully to both verify known targets and elucidate novel host factors required by toxins. Here, we show that this approach fails to identify host factors required for cell death due to βhc, a toxin required for GBS virulence. These data suggest that βhc may not require a host cell receptor for toxin function or may require a host receptor that is an essential gene and would not be identified using this screening strategy.}, }
@article {pmid35196219, year = {2022}, author = {Simon, DA and Tálas, A and Kulcsár, PI and Biczók, Z and Krausz, SL and Várady, G and Welker, E}, title = {PEAR, a flexible fluorescent reporter for the identification and enrichment of successfully prime edited cells.}, journal = {eLife}, volume = {11}, number = {}, pages = {}, pmid = {35196219}, issn = {2050-084X}, mesh = {Animals ; Bacteria ; *CRISPR-Cas Systems ; Fluorescence ; Gene Editing/*methods ; *Genes, Reporter ; Green Fluorescent Proteins/metabolism ; HEK293 Cells ; Humans ; }, abstract = {Prime editing is a recently developed CRISPR/Cas9 based gene engineering tool that allows the introduction of short insertions, deletions, and substitutions into the genome. However, the efficiency of prime editing, which typically achieves editing rates of around 10%-30%, has not matched its versatility. Here, we introduce the prime editor activity reporter (PEAR), a sensitive fluorescent tool for identifying single cells with prime editing activity. PEAR has no background fluorescence and specifically indicates prime editing events. Its design provides apparently unlimited flexibility for sequence variation along the entire length of the spacer sequence, making it uniquely suited for systematic investigation of sequence features that influence prime editing activity. The use of PEAR as an enrichment marker for prime editing can increase the edited population by up to 84%, thus significantly improving the applicability of prime editing for basic research and biotechnological applications.}, }
@article {pmid35196217, year = {2022}, author = {Donà, V and Ramette, A and Perreten, V}, title = {Comparative genomics of 26 complete circular genomes of 18 different serotypes of Actinobacillus pleuropneumoniae.}, journal = {Microbial genomics}, volume = {8}, number = {2}, pages = {}, pmid = {35196217}, issn = {2057-5858}, mesh = {*Actinobacillus pleuropneumoniae/genetics ; Animals ; Genomics/methods ; Lipopolysaccharides ; Multilocus Sequence Typing ; Serogroup ; Swine ; }, abstract = {Actinobacillus pleuropneumoniae is a Gram-negative, rod-shaped bacterium of the family Pasteurellaceae causing pig pleuropneumonia associated with great economic losses worldwide. Nineteen serotypes with distinctive lipopolysaccharide (LPS) and capsular (CPS) compositions have been described so far, yet complete circular genomes are publicly available only for the reference strains of serotypes 1, 4 and 5b, and for field strains of serotypes 1, 3, 7 and 8. We aimed to complete this picture by sequencing the reference strains of 17 different serotypes with the MinION sequencer (Oxford Nanopore Technologies, ONT) and on an Illumina HiSeq (Illumina) platform. We also included two field isolates of serotypes 2 and 3 that were PacBio- and MinION-sequenced, respectively. Genome assemblies were performed following two different strategies, i.e. PacBio- or ONT-only de novo assemblies polished with Illumina reads or a hybrid assembly by directly combining ONT and Illumina reads. Both methods proved successful in obtaining accurate circular genomes with comparable qualities. blast-based genome comparisons and core-genome phylogeny based on core genes, SNP typing and multi-locus sequence typing (cgMLST) of the 26 circular genomes indicated well-conserved genomes across the 18 different serotypes, differing mainly in phage insertions, and CPS, LPS and RTX-toxin clusters, which, consistently, encode serotype-specific antigens. We also identified small antibiotic resistance plasmids, and complete subtype I-F and subtype II-C CRISPR-Cas systems. Of note, highly similar clusters encoding all those serotype-specific traits were also found in other pathogenic and commensal Actinobacillus species. Taken together with the presence of transposable elements surrounding these loci, we speculate a dynamic intra- and interspecies exchange of such virulence-related factors by horizontal gene transfer. In conclusion, our comprehensive genomics analysis provides useful information for diagnostic test and vaccine development, but also for whole-genome-based epidemiological studies, as well as for the surveillance of the evolution of antibiotic resistance and virulence genes in A. pleuropneumoniae.}, }
@article {pmid35195982, year = {2022}, author = {Yan, X and Zhang, J and Jiang, Q and Jiao, D and Cheng, Y}, title = {Integration of the Ligase Chain Reaction with the CRISPR-Cas12a System for Homogeneous, Ultrasensitive, and Visual Detection of microRNA.}, journal = {Analytical chemistry}, volume = {94}, number = {9}, pages = {4119-4125}, doi = {10.1021/acs.analchem.2c00294}, pmid = {35195982}, issn = {1520-6882}, mesh = {*CRISPR-Cas Systems/genetics ; *Ligase Chain Reaction/methods ; *MicroRNAs/analysis/genetics ; Nucleic Acid Amplification Techniques/methods ; *RNA Probes/genetics ; RNA, Guide/genetics ; }, abstract = {The ligase chain reaction (LCR), as a classic nucleic acid amplification technique, is popular in the detection of DNA and RNA due to its simplicity, powerfulness, and high specificity. However, homogeneous and ultrasensitive LCR detection is still quite challenging. Herein, we integrate the LCR with a CRISPR-Cas12a system to greatly promote the application of the LCR in a homogeneous fashion. By employing microRNA as the model target, we design LCR probes with specific protospacer adjacent motif sequences and the guide RNA. Then, the LCR is initiated by target microRNA, and the LCR products specifically bind to the guide RNA to activate the Cas12a system, triggering secondary signal amplification to achieve ultrasensitive detection of microRNA without separation steps. Moreover, by virtue of a cationic conjugated polymer, microRNA can not only be visually detected by naked eyes but also be accurately quantified based on RGB ratio analysis of images with no need of sophisticated instruments. The method can quantify microRNA up to 4 orders of magnitude, and the determination limit is 0.4 aM, which is better than those of other reported studies using CRISPR-Cas12a and can be compared with that of the reverse-transcription polymerase chain reaction. This study demonstrates that the CRISPR-Cas12a system can greatly expand the application of the LCR for the homogeneous, ultrasensitive, and visual detection of microRNA, showing great potential in efficient nucleic acid detection and in vitro diagnosis.}, }
@article {pmid35195954, year = {2022}, author = {Aralis, Z and Rauch, JN and Audouard, M and Valois, E and Lach, RS and Solley, S and Baxter, NJ and Kosik, KS and Wilson, MZ and Acosta-Alvear, D and Arias, C}, title = {CREST, a Cas13-Based, Rugged, Equitable, Scalable Testing (CREST) for SARS-CoV-2 Detection in Patient Samples.}, journal = {Current protocols}, volume = {2}, number = {2}, pages = {e385}, doi = {10.1002/cpz1.385}, pmid = {35195954}, issn = {2691-1299}, mesh = {*COVID-19 ; CRISPR-Cas Systems ; Humans ; Nucleic Acid Amplification Techniques ; Pandemics ; *SARS-CoV-2 ; }, abstract = {The COVID-19 pandemic has taken a devastating human toll worldwide. The development of impactful guidelines and measures for controlling the COVID-19 pandemic requires continuous and widespread testing of suspected cases and their contacts through accurate, accessible, and reliable methods for SARS-CoV-2 detection. Here we describe a CRISPR-Cas13-based method for the detection of SARS-CoV-2. The assay is called CREST (Cas13-based, rugged, equitable, scalable testing), and is specific, sensitive, and highly accessible. As such, CREST may provide a low-cost and dependable alternative for SARS-CoV-2 surveillance. © 2022 Wiley Periodicals LLC. Basic Protocol: Cas13-ased detection of SARS-CoV-2 genetic material using a real-time PCR detection system Alternate Protocol: Cas13-based detection of SARS-CoV-2 genetic material using a fluorescence viewer Support Protocol 1: LwaCas13a purification Support Protocol 2: In vitro transcription of synthetic targets.}, }
@article {pmid35194830, year = {2022}, author = {Eerkens, AL and Vledder, A and van Rooij, N and Foijer, F and Nijman, HW and de Bruyn, M}, title = {Rapid and efficient generation of antigen-specific isogenic T cells from cryopreserved blood samples.}, journal = {Immunology and cell biology}, volume = {100}, number = {4}, pages = {285-295}, doi = {10.1111/imcb.12538}, pmid = {35194830}, issn = {1440-1711}, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Leukocytes, Mononuclear ; Mice ; }, abstract = {Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated gene editing has been leveraged for the modification of human and mouse T cells. However, limited experience is available on the application of CRISPR/Cas9 electroporation in cryopreserved T cells collected during clinical trials. To address this, we aimed to optimize a CRISPR/Cas9-mediated gene editing protocol compatible with peripheral blood mononuclear cells (PBMCs) samples routinely produced during clinical trials. PBMCs from healthy donors were used to generate knockout T-cell models for interferon-γ, Cbl proto-oncogene B (CBLB), Fas cell surface death receptor (Fas) and T-cell receptor (TCRαβ) genes. The effect of CRISPR/Cas9-mediated gene editing on T cells was evaluated using apoptosis assays, cytokine bead arrays and ex vivo and in vitro stimulation assays. Our results demonstrate that CRISPR/Cas9-mediated gene editing of ex vivo T cells is efficient and does not overtly affect T-cell viability. Cytokine release and T-cell proliferation were not affected in gene-edited T cells. Interestingly, memory T cells were more susceptible to CRISPR/Cas9 gene editing than naïve T cells. Ex vivo and in vitro stimulation with antigens resulted in equivalent antigen-specific T-cell responses in gene-edited and untouched control cells, making CRISPR/Cas9-mediated gene editing compatible with clinical antigen-specific T-cell activation and expansion assays. Here, we report an optimized protocol for rapid, viable and highly efficient genetic modification in ex vivo human antigen-specific T cells, for subsequent functional evaluation and/or expansion. Our platform extends CRISPR/Cas9-mediated gene editing for use in gold-standard clinically used immune-monitoring pipelines and serves as a starting point for development of analogous approaches, such as those including transcriptional activators and/or epigenetic modifiers.}, }
@article {pmid35194511, year = {2022}, author = {Dubey, AK and Kumar Gupta, V and Kujawska, M and Orive, G and Kim, NY and Li, CZ and Kumar Mishra, Y and Kaushik, A}, title = {Exploring nano-enabled CRISPR-Cas-powered strategies for efficient diagnostics and treatment of infectious diseases.}, journal = {Journal of nanostructure in chemistry}, volume = {}, number = {}, pages = {1-32}, pmid = {35194511}, issn = {2008-9244}, abstract = {Biomedical researchers have subsequently been inspired the development of new approaches for precisely changing an organism's genomic DNA in order to investigate customized diagnostics and therapeutics utilizing genetic engineering techniques. Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) is one such technique that has emerged as a safe, targeted, and effective pharmaceutical treatment against a wide range of disease-causing organisms, including bacteria, fungi, parasites, and viruses, as well as genetic abnormalities. The recent discovery of very flexible engineered nucleic acid binding proteins has changed the scientific area of genome editing in a revolutionary way. Since current genetic engineering technique relies on viral vectors, issues about immunogenicity, insertional oncogenesis, retention, and targeted delivery remain unanswered. The use of nanotechnology has the potential to improve the safety and efficacy of CRISPR/Cas9 component distribution by employing tailored polymeric nanoparticles. The combination of two (CRISPR/Cas9 and nanotechnology) offers the potential to open new therapeutic paths. Considering the benefits, demand, and constraints, the goal of this research is to acquire more about the biology of CRISPR technology, as well as aspects of selective and effective diagnostics and therapies for infectious illnesses and other metabolic disorders. This review advocated combining nanomedicine (nanomedicine) with a CRISPR/Cas enabled sensing system to perform early-stage diagnostics and selective therapy of specific infectious disorders. Such a Nano-CRISPR-powered nanomedicine and sensing system would allow for successful infectious illness control, even on a personal level. This comprehensive study also discusses the current obstacles and potential of the predicted technology.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40097-022-00472-7.}, }
@article {pmid35194225, year = {2022}, author = {Bouvenot, G}, title = {[Interfering RNA and antisense oligonucleotide treatments currently available in France: An update].}, journal = {Bulletin de l'Academie nationale de medecine}, volume = {206}, number = {4}, pages = {554-558}, doi = {10.1016/j.banm.2022.02.005}, pmid = {35194225}, issn = {0001-4079}, abstract = {The arrival of anti-Covid-19 RNA vaccines in 2020 should not obscure the fact that for several years we have already had treatments based on interfering RNA or antisense oligonucleotides in a number of rare diseases with a very poor prognosis such as transthyretin amyloidosis, acute hepatic porphyria, primary hyperoxaluria, spinal muscular atrophy or familial hyperchylomicronemia. If their performance, unlike that of vaccines, is for the moment only qualified as moderate therapeutic progress (moderate clinical added value) in the therapeutic strategies against these diseases, it should be taken into account that their initial evaluation was penalized by a certain number of unfavorable factors: trials of small numbers, therapeutic modalities to be refined, the lack of hindsight on their long-term effects but especially the choice of the moment of the initiation of the treatment in the natural evolution of the sickness. This choice is not trivial because it is hard to imagine that the products used could, beyond a simple stabilization of the disease installed, allow its regression as soon as certain lesions formed are irreversible. This is why their very early implementation, possibly based on genetic screening, is an avenue to be seized in the interest of patients. But, in the competitive context of innovations in the field, interfering RNAs and antisense oligonucleotides will have to reckon with gene therapy and genome editing using the CRISPR-Cas 9 technique.}, }
@article {pmid35194207, year = {2022}, author = {Anglada-Girotto, M and Handschin, G and Ortmayr, K and Campos, AI and Gillet, L and Manfredi, P and Mulholland, CV and Berney, M and Jenal, U and Picotti, P and Zampieri, M}, title = {Combining CRISPRi and metabolomics for functional annotation of compound libraries.}, journal = {Nature chemical biology}, volume = {18}, number = {5}, pages = {482-491}, pmid = {35194207}, issn = {1552-4469}, support = {866004/ERC_/European Research Council/International ; R21 AI133191/AI/NIAID NIH HHS/United States ; 866004//EC | EC Seventh Framework Programm | FP7 Ideas: European Research Council (FP7-IDEAS-ERC - Specific Programme: "Ideas" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013))/ ; AI133191//DH | NIHR | Health Services Research Programme (NIHR Health Services Research Programme)/ ; }, mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Escherichia coli/genetics/metabolism ; Humans ; Metabolomics/methods ; }, abstract = {Molecular profiling of small molecules offers invaluable insights into the function of compounds and allows for hypothesis generation about small-molecule direct targets and secondary effects. However, current profiling methods are limited in either the number of measurable parameters or throughput. Here we developed a multiplexed, unbiased framework that, by linking genetic to drug-induced changes in nearly a thousand metabolites, allows for high-throughput functional annotation of compound libraries in Escherichia coli. First, we generated a reference map of metabolic changes from CRISPR interference (CRISPRi) with 352 genes in all major essential biological processes. Next, on the basis of the comparison of genetic changes with 1,342 drug-induced metabolic changes, we made de novo predictions of compound functionality and revealed antibacterials with unconventional modes of action (MoAs). We show that our framework, combining dynamic gene silencing with metabolomics, can be adapted as a general strategy for comprehensive high-throughput analysis of compound functionality from bacteria to human cell lines.}, }
@article {pmid35194172, year = {2022}, author = {Katti, A and Diaz, BJ and Caragine, CM and Sanjana, NE and Dow, LE}, title = {CRISPR in cancer biology and therapy.}, journal = {Nature reviews. Cancer}, volume = {22}, number = {5}, pages = {259-279}, pmid = {35194172}, issn = {1474-1768}, mesh = {Biology ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; Genome ; Humans ; *Neoplasms/genetics/therapy ; }, abstract = {Over the past decade, CRISPR has become as much a verb as it is an acronym, transforming biomedical research and providing entirely new approaches for dissecting all facets of cell biology. In cancer research, CRISPR and related tools have offered a window into previously intractable problems in our understanding of cancer genetics, the noncoding genome and tumour heterogeneity, and provided new insights into therapeutic vulnerabilities. Here, we review the progress made in the development of CRISPR systems as a tool to study cancer, and the emerging adaptation of these technologies to improve diagnosis and treatment.}, }
@article {pmid35193353, year = {2022}, author = {Liu, X and Kang, X and Lei, C and Ren, W and Liu, C}, title = {Programming the trans-cleavage Activity of CRISPR-Cas13a by Single-Strand DNA Blocker and Its Biosensing Application.}, journal = {Analytical chemistry}, volume = {94}, number = {9}, pages = {3987-3996}, doi = {10.1021/acs.analchem.1c05124}, pmid = {35193353}, issn = {1520-6882}, mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; DNA, Single-Stranded ; }, abstract = {The precise and controllable programming of the trans-cleavage activity of the CRISPR-Cas13a systems is significant but challenging for fabricating high-performance biosensing systems toward various kinds of biomolecule targets. In this work, we have demonstrated that under a critical low Mg2+ concentration, a simple and short single-stranded DNA (ssDNA) probe free of any modification can efficiently prevent the assembly of crRNA and LwaCas13a only by partially binding with the crRNA repeat region, thereby blocking the trans-cleavage activity of the LwaCas13a system. Furthermore, we have demonstrated that the blocked trans-cleavage activity of the LwaCas13a system can be recovered by various kinds of biologically important substances as long as they could specifically release the blocker DNA from the crRNA in a target-responsive manner, providing a facile route for the quantification of diverse biomarkers such as enzymes, antigens/proteins, and exosomes. To the best of our knowledge, this is reported for the first time that a simple ssDNA can be employed as the switch element to control the crRNA structure and regulate the trans-cleavage activity of Cas13a, which has enriched the CRISPR-Cas13a sensing toolbox and will greatly expand its application scope.}, }
@article {pmid35192680, year = {2022}, author = {Jiang, C and Qian, M and Gocho, Y and Yang, W and Du, G and Shen, S and Yang, JJ and Zhang, H}, title = {Genome-wide CRISPR/Cas9 screening identifies determinant of panobinostat sensitivity in acute lymphoblastic leukemia.}, journal = {Blood advances}, volume = {6}, number = {8}, pages = {2496-2509}, pmid = {35192680}, issn = {2473-9537}, support = {P50 GM115279/GM/NIGMS NIH HHS/United States ; R01 GM118578/GM/NIGMS NIH HHS/United States ; }, mesh = {Apoptosis ; CRISPR-Cas Systems ; *Histones/metabolism ; Humans ; Panobinostat/pharmacology ; *Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy/genetics ; Sirtuin 1/genetics/metabolism/pharmacology ; United States ; }, abstract = {Epigenetic alterations, including histone acetylation, contribute to the malignant transformation of hematopoietic cells and disease progression, as well as the emergence of chemotherapy resistance. Targeting histone acetylation provides new strategies for the treatment of cancers. As a pan-histone deacetylase inhibitor, panobinostat has been approved by the US Food and Drug Administration for the treatment of multiple myeloma and has shown promising antileukemia effects in acute lymphoblastic leukemia (ALL). However, the underlying drug resistance mechanism in ALL remains largely unknown. Using genome-wide Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas)9 (CRISPR/Cas9) screening, we identified mitochondrial activity as the driver of panobinostat resistance in ALL. Mechanistically, ectopic SIRT1 expression activated mitochondrial activity and sensitized ALL to panobinostat through activating mitochondria-related apoptosis pathway. Meanwhile, the transcription level of SIRT1 was significantly associated with panobinostat sensitivity across diverse tumor types and thus could be a potential biomarker of panobinostat response in cancers. Our data suggest that patients with higher SIRT1 expression in cancer cells might benefit from panobinostat treatment, supporting the implementation of combinatorial therapy with SIRT1 or mitochondrial activators to overcome panobinostat resistance.}, }
@article {pmid35192362, year = {2022}, author = {Wei, T and Wen, X and Niu, C and An, S and Wang, D and Xi, Z and Wang, NN}, title = {Design of Acetohydroxyacid Synthase Herbicide-Resistant Germplasm through MB-QSAR and CRISPR/Cas9-Mediated Base-Editing Approaches.}, journal = {Journal of agricultural and food chemistry}, volume = {70}, number = {9}, pages = {2817-2824}, doi = {10.1021/acs.jafc.1c07180}, pmid = {35192362}, issn = {1520-5118}, mesh = {*Acetolactate Synthase/genetics/metabolism ; CRISPR-Cas Systems ; Herbicide Resistance/genetics ; *Herbicides/pharmacology ; Quantitative Structure-Activity Relationship ; }, abstract = {The development of herbicide-resistant germplasm is significant in solving the increasingly severe weed problem in crop fields. In this study, we, for the first time, rationally designed a predictable and effective approach to create herbicide-resistant germplasm by combining mutation-dependent biomacromolecular quantitative structure-activity relationship (MB-QSAR) and CRISPR/Cas9-mediated base-editing strategies. Our results showed that the homozygous P197F-G654D-G655S or P197F-G654N-G655S Arabidopsis plants exhibited high resistance to multiple acetohydroxyacid synthase-inhibiting herbicides, including chlorsulfuron, bispyribac-sodium, and flucarbazone-sodium. Additionally, the plants with the homozygous P197S mutant displayed increased susceptibility to bispyribac-sodium than the wild-type but more resistance to flumetsulam than other mutants. Besides, we found that the herbicide resistance levels of the gene-edited plants have a good correlation with MB-QSAR prediction.}, }
@article {pmid35191754, year = {2022}, author = {Barrangou, R}, title = {CRISPR Rewrites the Future of Medicine.}, journal = {The CRISPR journal}, volume = {5}, number = {1}, pages = {1}, doi = {10.1089/crispr.2022.29144.rba}, pmid = {35191754}, issn = {2573-1602}, mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; }, }
@article {pmid35191752, year = {2022}, author = {Moreb, EA and Lynch, MD}, title = {A Meta-Analysis of gRNA Library Screens Enables an Improved Understanding of the Impact of gRNA Folding and Structural Stability on CRISPR-Cas9 Activity.}, journal = {The CRISPR journal}, volume = {5}, number = {1}, pages = {146-154}, doi = {10.1089/crispr.2021.0084}, pmid = {35191752}, issn = {2573-1602}, mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing ; Gene Library ; *RNA, Guide/genetics ; }, abstract = {CRISPR systems are known to be inhibited by unwanted secondary structures that form within the guide RNA (gRNA). The minimum free energy of predicted secondary structures has been used in prediction algorithms. However, the types of structures as well as the degree to which a predicted structure can inhibit Cas9/gRNA activity is not well characterized. Here, we perform a meta-analysis of 39 published CRISPR-Cas9 data sets to understand better the role of secondary structures in inhibiting gRNA activity. We (1) identify two distinct inhibitory structures that can form, (2) measure the prevalence of these structures in existing gRNA library data sets, and (3) provide free energy cutoffs at which these structures become inhibitory. First, we show that hairpins that form within the targeting portion (spacer) of the gRNA, having a minimum free energy of <-5 kcal/mol, negatively impact gRNA activity. Second, we demonstrate that a longer hairpin can form between the spacer and the nexus portion of the gRNA scaffold. A duplex stability of this longer hairpin of <-15 kcal/mol negatively impacts gRNA activity. These cutoffs help to explain conflicting impacts of free energy values in different data sets, as well as provide a guideline for future gRNA designs.}, }
@article {pmid35191751, year = {2022}, author = {Cárdenas, P and Esherick, LY and Chambonnier, G and Dey, S and Turlo, CV and Nasamu, AS and Niles, JC}, title = {GeneTargeter: Automated In Silico Design for Genome Editing in the Malaria Parasite, Plasmodium falciparum.}, journal = {The CRISPR journal}, volume = {5}, number = {1}, pages = {155-164}, pmid = {35191751}, issn = {2573-1602}, support = {P30 ES002109/ES/NIEHS NIH HHS/United States ; T32 ES007020/ES/NIEHS NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; *Malaria, Falciparum/genetics/parasitology ; *Parasites/genetics ; Plasmodium falciparum/genetics ; }, abstract = {Functional characterization of the multitude of poorly described proteins in the human malarial pathogen, Plasmodium falciparum, requires tools to enable genome-scale perturbation studies. Here, we present GeneTargeter (genetargeter.mit.edu), a software tool for automating the design of homology-directed repair donor vectors to achieve gene knockouts, conditional knockdowns, and epitope tagging of P. falciparum genes. We demonstrate GeneTargeter-facilitated genome-scale design of six different types of knockout and conditional knockdown constructs for the P. falciparum genome and validate the computational design process experimentally with successful donor vector assembly and transfection. The software's modular nature accommodates arbitrary destination vectors and allows customizable designs that extend the genome manipulation outcomes attainable in Plasmodium and other organisms.}, }
@article {pmid35191750, year = {2022}, author = {Yoon, HH and Ye, S and Lim, S and Jo, A and Lee, H and Hong, F and Lee, SE and Oh, SJ and Kim, NR and Kim, K and Kim, BJ and Kim, H and Lee, CJ and Nam, MH and Hur, JW and Jeon, SR}, title = {CRISPR-Cas9 Gene Editing Protects from the A53T-SNCA Overexpression-Induced Pathology of Parkinson's Disease In Vivo.}, journal = {The CRISPR journal}, volume = {5}, number = {1}, pages = {95-108}, doi = {10.1089/crispr.2021.0025}, pmid = {35191750}, issn = {2573-1602}, mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Mutation ; *Parkinson Disease/genetics/metabolism/pathology/therapy ; Rats ; *alpha-Synuclein/genetics/metabolism ; }, abstract = {Mutations in specific genes, including synuclein alpha (SNCA) that encodes the α-synuclein protein, are known to be risk factors for sporadic Parkinson's disease (PD), as well as critical factors for familial PD. In particular, A53T-mutated SNCA (A53T-SNCA) is a well-studied familial pathologic mutation in PD. However, techniques for deletion of the mutated SNCA gene in vivo have not been developed. Here, we used the CRISPR-Cas9 system to delete A53T-SNCA in vitro as well as in vivo. Adeno-associated virus carrying SaCas9-KKH with a single-guide RNA targeting A53T-SNCA significantly reduced A53T-SNCA expression levels in vitro. Furthermore, we tested its therapeutic potential in vivo in a viral A53T-SNCA-overexpressing rat model of PD. Gene deletion of A53T-SNCA significantly rescued the overexpression of α-synuclein, reactive microgliosis, dopaminergic neurodegeneration, and parkinsonian motor symptoms. Our findings propose CRISPR-Cas9 system as a potential prevention strategy for A53T-SNCA-specific PD.}, }
@article {pmid35191749, year = {2022}, author = {Miccio, A}, title = {CRISPR's Path to the Clinic.}, journal = {The CRISPR journal}, volume = {5}, number = {1}, pages = {2-3}, doi = {10.1089/crispr.2022.29143.am}, pmid = {35191749}, issn = {2573-1602}, mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; }, }
@article {pmid35190679, year = {2022}, author = {Sago, CD and Lokugamage, MP and Loughrey, D and Lindsay, KE and Hincapie, R and Krupczak, BR and Kalathoor, S and Sato, M and Echeverri, ES and Fitzgerald, JP and Gan, Z and Gamboa, L and Paunovska, K and Sanhueza, CA and Hatit, MZC and Finn, MG and Santangelo, PJ and Dahlman, JE}, title = {Augmented lipid-nanoparticle-mediated in vivo genome editing in the lungs and spleen by disrupting Cas9 activity in the liver.}, journal = {Nature biomedical engineering}, volume = {6}, number = {2}, pages = {157-167}, pmid = {35190679}, issn = {2157-846X}, mesh = {Animals ; Antigens, CD ; CRISPR-Cas Systems ; Cell Adhesion Molecules/genetics ; Endothelial Cells ; *Gene Editing ; Lipids/chemistry ; Liposomes ; Liver ; Lung ; Mice ; *Nanoparticles/chemistry ; Spleen ; }, abstract = {Systemically delivered lipid nanoparticles are preferentially taken up by hepatocytes. This hinders the development of effective, non-viral means of editing genes in tissues other than the liver. Here we show that lipid-nanoparticle-mediated gene editing in the lung and spleen of adult mice can be enhanced by reducing Cas9-mediated insertions and deletions in hepatocytes via oligonucleotides disrupting the secondary structure of single-guide RNAs (sgRNAs) and also via their combination with short interfering RNA (siRNA) targeting Cas9 messenger RNA (mRNA). In SpCas9 mice with acute lung inflammation, the systemic delivery of an oligonucleotide inhibiting an sgRNA targeting the intercellular adhesion molecule 2 (ICAM-2), followed by the delivery of the sgRNA, reduced the fraction of ICAM-2 indels in hepatocytes and increased that in lung endothelial cells. In wild-type mice, the lipid-nanoparticle-mediated delivery of an inhibitory oligonucleotide, followed by the delivery of Cas9-degrading siRNA and then by Cas9 mRNA and sgRNA, reduced the fraction of ICAM-2 indels in hepatocytes but not in splenic endothelial cells. Inhibitory oligonucleotides and siRNAs could be used to modulate the cell-type specificity of Cas9 therapies.}, }
@article {pmid35190677, year = {2022}, author = {Dilliard, SA and Siegwart, DJ}, title = {Disrupting off-target Cas9 activity in the liver.}, journal = {Nature biomedical engineering}, volume = {6}, number = {2}, pages = {106-107}, pmid = {35190677}, issn = {2157-846X}, mesh = {*CRISPR-Cas Systems ; Gene Editing ; Liver ; *RNA, Guide ; }, }
@article {pmid35189910, year = {2022}, author = {Wang, SW and Gao, C and Zheng, YM and Yi, L and Lu, JC and Huang, XY and Cai, JB and Zhang, PF and Cui, YH and Ke, AW}, title = {Current applications and future perspective of CRISPR/Cas9 gene editing in cancer.}, journal = {Molecular cancer}, volume = {21}, number = {1}, pages = {57}, pmid = {35189910}, issn = {1476-4598}, mesh = {Animals ; CRISPR-Cas Systems ; *Gene Editing/methods ; Genomics ; Humans ; *Neoplasms/genetics/therapy ; Oncogenes ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) system provides adaptive immunity against plasmids and phages in prokaryotes. This system inspires the development of a powerful genome engineering tool, the CRISPR/CRISPR-associated nuclease 9 (CRISPR/Cas9) genome editing system. Due to its high efficiency and precision, the CRISPR/Cas9 technique has been employed to explore the functions of cancer-related genes, establish tumor-bearing animal models and probe drug targets, vastly increasing our understanding of cancer genomics. Here, we review current status of CRISPR/Cas9 gene editing technology in oncological research. We first explain the basic principles of CRISPR/Cas9 gene editing and introduce several new CRISPR-based gene editing modes. We next detail the rapid progress of CRISPR screening in revealing tumorigenesis, metastasis, and drug resistance mechanisms. In addition, we introduce CRISPR/Cas9 system delivery vectors and finally demonstrate the potential of CRISPR/Cas9 engineering to enhance the effect of adoptive T cell therapy (ACT) and reduce adverse reactions.}, }
@article {pmid35189535, year = {2022}, author = {Liang, Y and Lin, H and Zou, L and Deng, X and Tang, S}, title = {Rapid detection and tracking of Omicron variant of SARS-CoV-2 using CRISPR-Cas12a-based assay.}, journal = {Biosensors &amp; bioelectronics}, volume = {205}, number = {}, pages = {114098}, doi = {10.1016/j.bios.2022.114098}, pmid = {35189535}, issn = {1873-4235}, mesh = {*Biosensing Techniques ; *COVID-19/diagnosis ; CRISPR-Cas Systems/genetics ; Humans ; SARS-CoV-2/genetics ; }, abstract = {BACKGROUND: The newly emerged SARS-CoV-2 variant of concern (VOC) Omicron is spreading quickly worldwide, which manifests an urgent need of simple and rapid assay to detect and diagnose Omicron infection and track its spread.<br><br>METHODS: To design allele-specific CRISPR RNAs (crRNAs) targeting the signature mutations in the spike protein of Omicron variant, and to develop a CRISPR-Cas12a-based assay to specifically detect Omicron variant.<br><br>RESULTS: Our system showed a low limit of detection of 2 copies per reaction for the plasmid DNA of Omicron variant, and could readily detect Omicron variant in 5 laboratory-confirmed clinical samples and distinguish them from 57 SARS-CoV-2 positive clinical samples (4 virus isolates and 53 oropharyngeal swab specimens) infected with wild-type (N = 8) and the variants of Alpha (N = 17), Beta (N = 17) and Delta (N = 15). The testing results could be measured by fluorescent detector or judged by naked eyes. In addition, no cross-reaction was observed when detecting 16 clinical samples infected with 9 common respiratory pathogens.<br><br>CONCLUSIONS: The rapid assay could be easily set up in laboratories already conducting SARS-CoV-2 nucleic acid amplification tests and implemented routinely in resource-limited settings to monitor and track the spread of Omicron variant.}, }
@article {pmid35188655, year = {2022}, author = {Kelterborn, S and Boehning, F and Sizova, I and Baidukova, O and Evers, H and Hegemann, P}, title = {Gene Editing in Green Alga Chlamydomonas reinhardtii via CRISPR-Cas9 Ribonucleoproteins.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2379}, number = {}, pages = {45-65}, pmid = {35188655}, issn = {1940-6029}, mesh = {CRISPR-Cas Systems/genetics ; *Chlamydomonas reinhardtii/genetics ; Electroporation/methods ; *Gene Editing/methods ; Ribonucleoproteins/genetics ; }, abstract = {With the establishment of the CRISPR-Cas9 molecular tool as a DNA editing system in 2012, the handling of gene editing experiments was strongly facilitated pushing reverse genetics approaches forward in many organisms. These new gene editing technologies also drastically increased the possibilities for design-driven synthetic biology. Here, we describe a protocol for gene editing in the green algae Chlamydomonas reinhardtii using preassembled CRISPR-Cas9 ribonucleoproteins.The three sections of the protocol guide through a complete gene editing experiment, starting with the experimental design and the choice of suitable CRISPR target sites and how to perform a Cas9 in vitro test digestion. The second part covers the transformation of algal cells with Cas9 RNPs using electroporation. In the last part, the PCR-based screening for mutants and isolation of clones is explained.}, }
@article {pmid35188654, year = {2022}, author = {Vazquez-Vilar, M and Juarez, P and Bernabé-Orts, JM and Orzaez, D}, title = {Design of Multiplexing CRISPR/Cas9 Constructs for Plant Genome Engineering Using the GoldenBraid DNA Assembly Standard.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2379}, number = {}, pages = {27-44}, pmid = {35188654}, issn = {1940-6029}, mesh = {*CRISPR-Cas Systems/genetics ; DNA ; *Gene Editing/methods ; Genome, Plant/genetics ; RNA, Guide/genetics ; }, abstract = {Due to the huge potential of CRISPR/Cas9 for synthetic biology and genome engineering, many plant researchers are adopting this technology in their laboratories. CRISPR/Cas9 allows multiplexing of guide RNAs (gRNAs), therefore targeting several loci in the genome simultaneously. However, making DNA constructs for this purpose is not always straightforward for first-time users. Here we show how to make multiplex CRISPR/Cas9 constructs using the GoldenBraid (GB) DNA assembly system. As an example, we create a polycistronic gRNA construct that guides a dead version of Cas9 to three different positions of the nopaline synthase promoter, leading to transcriptional repression. After a description of the reagents, the protocol describes step-by-step the considerations for DNA target selection and the molecular cloning process of the final T-DNA construct as well as its testing by transient expression in Nicotiana benthamiana leaves along with a reporter construct for luciferase expression.}, }
@article {pmid35188653, year = {2022}, author = {Dudley, QM and Raitskin, O and Patron, NJ}, title = {Cas9-Mediated Targeted Mutagenesis in Plants.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2379}, number = {}, pages = {1-26}, pmid = {35188653}, issn = {1940-6029}, mesh = {*CRISPR-Cas Systems ; *Genome, Plant ; Mutagenesis ; Plants, Genetically Modified/genetics ; Protoplasts/metabolism ; }, abstract = {Genome engineering technologies enable targeted mutations to be induced at almost any location in plant genomes. In particular, Cas9 nucleases use easily recoded RNA guides to target user-defined sequences and generate double-stranded breaks (DSB) that are then repaired by the cell's endogenous repair mechanisms. Incorrect repair results in mutations at the target. When the targets are in coding sequences, this often results in loss-of-function mutations. In this chapter, we describe a method to rapidly design and assemble RNA-guided Cas9 constructs for plants and test their ability to induce mutations at their intended targets in rapid assays using both Agrobacterium-mediated transient expression and PEG-mediated DNA delivery to protoplasts, the latter of which can be adapted to a wide range of plant species. We describe a PCR-based method for detecting mutagenesis and outline the steps required to segregate the Cas9 transgene from the targeted mutation to enable the production of transgene-free mutated plants. These techniques are amenable to a range of plant species and should accelerate the application of Cas-9-mediated genome engineering for basic plant science as well as crop development.}, }
@article {pmid35188577, year = {2022}, author = {Song, G and Zhang, F and Tian, C and Gao, X and Zhu, X and Fan, D and Tian, Y}, title = {Discovery of potent and versatile CRISPR-Cas9 inhibitors engineered for chemically controllable genome editing.}, journal = {Nucleic acids research}, volume = {50}, number = {5}, pages = {2836-2853}, pmid = {35188577}, issn = {1362-4962}, mesh = {Bacteriophages/genetics/metabolism ; *CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; Interspersed Repetitive Sequences ; Streptococcus/*genetics/virology ; }, abstract = {Anti-CRISPR (Acr) proteins are encoded by many mobile genetic elements (MGEs) such as phages and plasmids to combat CRISPR-Cas adaptive immune systems employed by prokaryotes, which provide powerful tools for CRISPR-Cas-based applications. Here, we discovered nine distinct type II-A anti-CRISPR (AcrIIA24-32) families from Streptococcus MGEs and found that most Acrs can potently inhibit type II-A Cas9 orthologs from Streptococcus (SpyCas9, St1Cas9 or St3Cas9) in bacterial and human cells. Among these Acrs, AcrIIA26, AcrIIA27, AcrIIA30 and AcrIIA31 are able to block Cas9 binding to DNA, while AcrIIA24 abrogates DNA cleavage by Cas9. Notably, AcrIIA25.1 and AcrIIA32.1 can inhibit both DNA binding and DNA cleavage activities of SpyCas9, exhibiting unique anti-CRISPR characteristics. Importantly, we developed several chemically inducible anti-CRISPR variants based on AcrIIA25.1 and AcrIIA32.1 by comprising hybrids of Acr protein and the 4-hydroxytamoxifen-responsive intein, which enabled post-translational control of CRISPR-Cas9-mediated genome editing in human cells. Taken together, our work expands the diversity of type II-A anti-CRISPR families and the toolbox of Acr proteins for the chemically inducible control of Cas9-based applications.}, }
@article {pmid35187887, year = {2022}, author = {Li, X and Sun, B and Qian, H and Ma, J and Paolino, M and Zhang, Z}, title = {A high-efficiency and versatile CRISPR/Cas9-mediated HDR-based biallelic editing system.}, journal = {Journal of Zhejiang University. Science. B}, volume = {23}, number = {2}, pages = {141-152}, pmid = {35187887}, issn = {1862-1783}, support = {2018ZX08010-09B//the National Science and Technology Major Project of China/ ; NE 2016-04458//the Swedish Research Council/ ; M21/17//the Ragnar Söderberg Foundation/ ; }, mesh = {Alleles ; *CRISPR-Cas Systems ; DNA End-Joining Repair ; Gene Editing/methods ; *Recombinational DNA Repair ; }, abstract = {Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9), the third-generation genome editing tool, has been favored because of its high efficiency and clear system composition. In this technology, the introduced double-strand breaks (DSBs) are mainly repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways. The high-fidelity HDR pathway is used for genome modification, which can introduce artificially controllable insertions, deletions, or substitutions carried by the donor templates. Although high-level knock-out can be easily achieved by NHEJ, accurate HDR-mediated knock-in remains a technical challenge. In most circumstances, although both alleles are broken by endonucleases, only one can be repaired by HDR, and the other one is usually recombined by NHEJ. For gene function studies or disease model establishment, biallelic editing to generate homozygous cell lines and homozygotes is needed to ensure consistent phenotypes. Thus, there is an urgent need for an efficient biallelic editing system. Here, we developed three pairs of integrated selection systems, where each of the two selection cassettes contained one drug-screening gene an