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Bibliography on: CRISPR-Cas

The Electronic Scholarly Publishing Project: Providing world-wide, free access to classic scientific papers and other scholarly materials, since 1993.


ESP: PubMed Auto Bibliography 21 Mar 2023 at 01:42 Created: 


Clustered regularly interspaced short palindromic repeats (CRISPR, pronounced crisper) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to foreign DNA (e.g a virus or plasmid). The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages, and provides a form of acquired immunity. CRISPR associated proteins (Cas) use the CRISPR spacers to recognize and cut these exogenous genetic elements in a manner analogous to RNA interference in eukaryotic organisms. CRISPRs are found in approximately 40% of sequenced bacterial genomes and 90% of sequenced archaea. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added. The Cas9-gRNA complex corresponds with the CAS III crRNA complex in the above diagram. CRISPR/Cas genome editing techniques have many potential applications, including altering the germline of humans, animals, and food crops. The use of CRISPR Cas9-gRNA complex for genome editing was the AAAS's choice for breakthrough of the year in 2015.

Created with PubMed® Query: ( "CRISPR.CAS" OR "crispr/cas" ) NOT pmcbook NOT ispreviousversion

Citations The Papers (from PubMed®)


RevDate: 2023-03-20

Gu B, Zhuo C, Xu X, et al (2023)

Editorial: Molecular diagnostics for infectious diseases: Novel approaches, clinical applications and future challenges.

Frontiers in microbiology, 14:1153827.

RevDate: 2023-03-20

Xiao H, Hu J, Huang C, et al (2023)

CRISPR techniques and potential for the detection and discrimination of SARS-CoV-2 variants of concern.

Trends in analytical chemistry : TRAC, 161:117000.

The continuing evolution of the SARS-CoV-2 virus has led to the emergence of many variants, including variants of concern (VOCs). CRISPR-Cas systems have been used to develop techniques for the detection of variants. These techniques have focused on the detection of variant-specific mutations in the spike protein gene of SARS-CoV-2. These sequences mostly carry single-nucleotide mutations and are difficult to differentiate using a single CRISPR-based assay. Here we discuss the specificity of the Cas9, Cas12, and Cas13 systems, important considerations of mutation sites, design of guide RNA, and recent progress in CRISPR-based assays for SARS-CoV-2 variants. Strategies for discriminating single-nucleotide mutations include optimizing the position of mismatches, modifying nucleotides in the guide RNA, and using two guide RNAs to recognize the specific mutation sequence and a conservative sequence. Further research is needed to confront challenges in the detection and differentiation of variants and sublineages of SARS-CoV-2 in clinical diagnostic and point-of-care applications.

RevDate: 2023-03-19

Marino ND (2023)

Phage against the machine: discovery and mechanism of type V anti-CRISPRs.

Journal of molecular biology pii:S0022-2836(23)00110-9 [Epub ahead of print].

The discovery of diverse bacterial CRISPR-Cas systems has reignited interest in understanding bacterial defense pathways while yielding exciting new tools for genome editing. CRISPR-Cas systems are widely distributed in prokaryotes, found in 40% of bacteria and 90% of archaea, where they function as adaptive immune systems against bacterial viruses (phage) and other mobile genetic elements. In turn, phage have evolved inhibitors, called anti-CRISPR proteins, to prevent targeting. Type V CRISPR-Cas12 systems have emerged as a particularly exciting arena in this co-evolutionary arms race. Type V anti-CRISPRs have highly diverse and novel mechanisms of action, some of which appear to be unusually potent or widespread. In this review, we discuss the discovery and mechanism of these anti-CRISPRs as well as future areas for exploration.

RevDate: 2023-03-18

Bhatia S, Pooja , SK Yadav (2023)

CRISPR-Cas for genome editing: Classification, mechanism, designing and applications.

International journal of biological macromolecules pii:S0141-8130(23)00948-0 [Epub ahead of print].

Clustered regularly interspersed short pallindromic repeats (CRISPR) and CRISPR associated proteins (Cas) system (CRISPR-Cas) came into light as prokaryotic defence mechanism for adaptive immune response. CRISPR-Cas works by integrating short sequences of the target genome (spacers) into the CRISPR locus. The locus containing spacers interspersed repeats is further expressed into small guide CRISPR RNA (crRNA) which is then deployed by the Cas proteins to evade the target genome. Based on the Cas proteins CRISPR-Cas is classified according to polythetic system of classification. The characteristic of the CRISPR-Cas9 system to target DNA sequences using programmable RNAs has opened new arenas due to which today CRISPR-Cas has evolved as cutting end technique in the field of genome editing. Here, we discuss about the evolution of CRISPR, its classification and various Cas systems including the designing and molecular mechanism of CRISPR-Cas. Applications of CRISPR-Cas as a genome editing tools are also highlighted in the areas such as agriculture, and anticancer therapy. Briefly discuss the role of CRISPR and its Cas systems in the diagnosis of COVID-19 and its possible preventive measures. The challenges in existing CRISP-Cas technologies and their potential solutions are also discussed briefly.

RevDate: 2023-03-17

Rostain W, Grebert T, Vyhovskyi D, et al (2023)

Cas9 off-target binding to the promoter of bacterial genes leads to silencing and toxicity.

Nucleic acids research pii:7079636 [Epub ahead of print].

Genetic tools derived from the Cas9 RNA-guided nuclease are providing essential capabilities to study and engineer bacteria. While the importance of off-target effects was noted early in Cas9's application to mammalian cells, off-target cleavage by Cas9 in bacterial genomes is easily avoided due to their smaller size. Despite this, several studies have reported experimental setups in which Cas9 expression was toxic, even when using the catalytic dead variant of Cas9 (dCas9). Specifically, dCas9 was shown to be toxic when in complex with guide RNAs sharing specific PAM (protospacer adjacent motif)-proximal sequence motifs. Here, we demonstrate that this toxicity is caused by off-target binding of Cas9 to the promoter of essential genes, with silencing of off-target genes occurring with as little as 4 nt of identity in the PAM-proximal sequence. Screens performed in various strains of Escherichia coli and other enterobacteria show that the nature of toxic guide RNAs changes together with the evolution of sequences at off-target positions. These results highlight the potential for Cas9 to bind to hundreds of off-target positions in bacterial genomes, leading to undesired effects. This phenomenon must be considered in the design and interpretation of CRISPR-Cas experiments in bacteria.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Wu ZS, Gao Y, Du YT, et al (2023)

The protocol of tagging endogenous proteins with fluorescent tags using CRISPR-Cas9 genome editing.

Yi chuan = Hereditas, 45(2):165-175.

The currently widely used CRISPR-Cas9 genome editing technology enables the editing of target genes (knock-out or knock-in) with high accuracy and efficiency. Guided by the small guide RNA, the Cas9 nuclease induces a DNA double-strand break at the targeted genomic locus. The DNA double-strand break can be repaired by the homology-directed repair pathway in the presence of a repair template. With the repair template containing the coding sequence of a fluorescent tag, the targeted gene can be inserted with the sequence of a fluorescent tag at the designed position. The genome editing mediated labeling of endogenous proteins with fluorescent tags avoids the potential artifacts caused by gene overexpression and substantially improves the reproductivity of imaging experiments. This protocol focuses on creating mammalian cell lines with endogenous proteins tagged with fluorescent proteins or self-labeling protein tags using CRISPR-Cas9 genome editing.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Liu MZ, Wang LR, Li YM, et al (2023)

Generation of genetically modified rat models via the CRISPR/Cas9 technology.

Yi chuan = Hereditas, 45(1):78-87.

The RNA-guided CRISPR/Cas9 genomic editing system consists of a single guide RNA (sgRNA) and a Cas9 nuclease. The two components form a complex in cells and target the genomic loci complementary to the sgRNA. The Cas9 nuclease cleaves the target site creating a double stranded DNA break (DSB). In mammalian cells, DSBs are often repaired via error prone non-homologous end joining (NHEJ) or via homology directed repair (HDR) with the presence of donor DNA templates. Micro-injection of the CRISPR/Cas9 system into the rat embryos enables generation of genetically modified rat models. Here, we describe a detailed protocol for creating gene knockout or knockin rat models via the CRISPR/Cas9 technology.

RevDate: 2023-03-20
CmpDate: 2023-03-20

D'Antonio L, Fieni C, Ciummo SL, et al (2023)

Inactivation of interleukin-30 in colon cancer stem cells via CRISPR/Cas9 genome editing inhibits their oncogenicity and improves host survival.

Journal for immunotherapy of cancer, 11(3):.

BACKGROUND: Progression of colorectal cancer (CRC), a leading cause of cancer-related death worldwide, is driven by colorectal cancer stem cells (CR-CSCs), which are regulated by endogenous and microenvironmental signals. Interleukin (IL)-30 has proven to be crucial for CSC viability and tumor progression. Whether it is involved in CRC tumorigenesis and impacts clinical behavior is unknown.

METHODS: IL30 production and functions, in stem and non-stem CRC cells, were determined by western blot, immunoelectron microscopy, flow cytometry, cell viability and sphere formation assays. CRISPR/Cas9-mediated deletion of the IL30 gene, RNA-Seq and implantation of IL30 gene transfected or deleted CR-CSCs in NSG mice allowed to investigate IL30's role in CRC oncogenesis. Bioinformatics and immunopathology of CRC samples highlighted the clinical implications.

RESULTS: We demonstrated that both CR-CSCs and CRC cells express membrane-anchored IL30 that regulates their self-renewal, via WNT5A and RAB33A, and/or proliferation and migration, primarily by upregulating CXCR4 via STAT3, which are suppressed by IL30 gene deletion, along with WNT and RAS pathways. Deletion of IL30 gene downregulates the expression of proteases, such as MMP2 and MMP13, chemokine receptors, mostly CCR7, CCR3 and CXCR4, and growth and inflammatory mediators, including ANGPT2, CXCL10, EPO, IGF1 and EGF. These factors contribute to IL30-driven CR-CSC and CRC cell expansion, which is abrogated by their selective blockade. IL30 gene deleted CR-CSCs displayed reduced tumorigenicity and gave rise to slow-growing and low metastatic tumors in 80% of mice, which survived much longer than controls. Bioinformatics and CIBERSORTx of the 'Colorectal Adenocarcinoma TCGA Nature 2012' collection, and morphometric assessment of IL30 expression in clinical CRC samples revealed that the lack of IL30 in CRC and infiltrating leucocytes correlates with prolonged overall survival.

CONCLUSIONS: IL30 is a new CRC driver, since its inactivation, which disables oncogenic pathways and multiple autocrine loops, inhibits CR-CSC tumorigenicity and metastatic ability. The development of CRISPR/Cas9-mediated targeting of IL30 could improve the current therapeutic landscape of CRC.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Wang R, Zhang R, Khodaverdian A, et al (2023)

Theoretical guarantees for phylogeny inference from single-cell lineage tracing.

Proceedings of the National Academy of Sciences of the United States of America, 120(12):e2203352120.

Lineage-tracing technologies based on Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9 (CRISPR-Cas9) genome editing have emerged as a powerful tool for investigating development in single-cell contexts, but exact reconstruction of the underlying clonal relationships in experiment is complicated by features of the data. These complications are functions of the experimental parameters in these systems, such as the Cas9 cutting rate, the diversity of indel outcomes, and the rate of missing data. In this paper, we develop two theoretically grounded algorithms for the reconstruction of the underlying single-cell phylogenetic tree as well as asymptotic bounds for the number of recording sites necessary for exact recapitulation of the ground truth phylogeny at high probability. In doing so, we explore the relationship between the problem difficulty and the experimental parameters, with implications for experimental design. Lastly, we provide simulations showing the empirical performance of these algorithms and showing that the trends in the asymptotic bounds hold empirically. Overall, this work provides a theoretical analysis of phylogenetic reconstruction in single-cell CRISPR-Cas9 lineage-tracing technologies.

RevDate: 2023-03-18

Meng X, Wu TG, Lou QY, et al (2023)

Optimization of CRISPR-Cas system for clinical cancer therapy.

Bioengineering & translational medicine, 8(2):e10474.

Cancer is a genetic disease caused by alterations in genome and epigenome and is one of the leading causes for death worldwide. The exploration of disease development and therapeutic strategies at the genetic level have become the key to the treatment of cancer and other genetic diseases. The functional analysis of genes and mutations has been slow and laborious. Therefore, there is an urgent need for alternative approaches to improve the current status of cancer research. Gene editing technologies provide technical support for efficient gene disruption and modification in vivo and in vitro, in particular the use of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems. Currently, the applications of CRISPR-Cas systems in cancer rely on different Cas effector proteins and the design of guide RNAs. Furthermore, effective vector delivery must be met for the CRISPR-Cas systems to enter human clinical trials. In this review article, we describe the mechanism of the CRISPR-Cas systems and highlight the applications of class II Cas effector proteins. We also propose a synthetic biology approach to modify the CRISPR-Cas systems, and summarize various delivery approaches facilitating the clinical application of the CRISPR-Cas systems. By modifying the CRISPR-Cas system and optimizing its in vivo delivery, promising and effective treatments for cancers using the CRISPR-Cas system are emerging.

RevDate: 2023-03-18

Khambhati K, Bhattacharjee G, Gohil N, et al (2023)

Phage engineering and phage-assisted CRISPR-Cas delivery to combat multidrug-resistant pathogens.

Bioengineering & translational medicine, 8(2):e10381.

Antibiotic resistance ranks among the top threats to humanity. Due to the frequent use of antibiotics, society is facing a high prevalence of multidrug resistant pathogens, which have managed to evolve mechanisms that help them evade the last line of therapeutics. An alternative to antibiotics could involve the use of bacteriophages (phages), which are the natural predators of bacterial cells. In earlier times, phages were implemented as therapeutic agents for a century but were mainly replaced with antibiotics, and considering the menace of antimicrobial resistance, it might again become of interest due to the increasing threat of antibiotic resistance among pathogens. The current understanding of phage biology and clustered regularly interspaced short palindromic repeats (CRISPR) assisted phage genome engineering techniques have facilitated to generate phage variants with unique therapeutic values. In this review, we briefly explain strategies to engineer bacteriophages. Next, we highlight the literature supporting CRISPR-Cas9-assisted phage engineering for effective and more specific targeting of bacterial pathogens. Lastly, we discuss techniques that either help to increase the fitness, specificity, or lytic ability of bacteriophages to control an infection.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Chen H, Li B, Shi S, et al (2023)

Au-Fe3O4 nanozyme coupled with CRISPR-Cas12a for sensitive and visual antibiotic resistance diagnosing.

Analytica chimica acta, 1251:341014.

The accumulation and spread of antibiotic resistance bacteria (ARB) in the environment may accelerate the formation of superbugs and seriously threaten the health of all living beings. The timeliness and accurate diagnosing of antibiotic resistance is essential to controlling the propagation of superbugs in the environment and formulating effective public health management programs. Herein, we developed a speedy, sensitive, accurate, and user-friendly colorimetric assay for antibiotic resistance, via a synergistic combination of the peroxidase-like property of the Au-Fe3O4 nanozyme and the specific gene identification capability of the CRISPR-Cas12a. Once the CRISPR-Cas12a system recognizes a target resistance gene, it activates its trans-cleavage activity and subsequently releases the Au-Fe3O4 nanozymes, which oxidizes the 3,3,5,5-tetramethylbenzidine (TMB) with color change from transparent to blue. The diagnosing signals could be captured and analyzed by a smartphone. This method detected kanamycin-resistance genes, ampicillin-resistance genes, and chloramphenicol-resistance genes by simple operation steps with high sensitivity (<0.1 CFU μL[-1]) and speediness (<1 h). This approach may prove easy for the accurate and sensitive diagnosis of the ARGs or ARB in the field, thus surveilling and controlling the microbial water quality flexibly and efficiently.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Niu C, Liu J, Xing X, et al (2023)

CRISPR-Cas12a-assisted elimination of the non-specific signal from non-specific amplification in the Exponential Amplification Reaction.

Analytica chimica acta, 1251:340998.

Non-specific amplification is a major problem in nucleic acid amplification resulting in false-positive results, especially for exponential amplification reactions (EXPAR). Although efforts were made to suppress the influence of non-specific amplification, such as chemical blocking of the template's 3'-ends and sequence-independent weakening of template-template interactions, it is still a common problem in many conventional EXPAR reactions. In this study, we propose a novel strategy to eliminate the non-specific signal from non-specific amplification by integrating the CRISPR-Cas12a system into two-templates EXPAR. An EXPAR-Cas12a strategy named EXPCas was developed, where the Cas12a system acted as a filter to filter out non-specific amplificons in EXPAR, suppressing and eliminating the influence of non-specific amplification. As a result, the signal-to-background ratio was improved from 1.3 to 15.4 using this method. With microRNA-21 (miRNA-21) as a target, the detection can be finished in 40 min with a LOD of 103 fM and no non-specific amplification was observed.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Yin Z, Su R, Ge L, et al (2023)

Single-cell resolution reveals RalA GTPase expanding hematopoietic stem cells and facilitating of BCR-ABL1-driven leukemogenesis in a CRISPR/Cas9 gene editing mouse model.

International journal of biological sciences, 19(4):1211-1227.

BCR-ABL oncogene-mediated Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) is suggested to originate from leukemic stem cells (LSCs); however, factors regulating self-renewal of LSC and normal hematopoietic stem cells (HSCs) are largely unclear. Here, we show that RalA, a small GTPase in the Ras downstream signaling pathway, has a critical effect on regulating the self-renewal of LSCs and HSCs. A RalA knock-in mouse model (RalA[Rosa26-Tg/+]) was initially constructed on the basis of the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) assay to analyze normal hematopoietic differentiation frequency using single-cell resolution and flow cytometry. RalA overexpression promoted cell cycle progression and increased the frequency of granulocyte-monocyte progenitors (GMPs), HSCs and multipotent progenitors (MPPs). The uniform manifold approximation and projection (UMAP) plot revealed heterogeneities in HSCs and progenitor cells (HSPCs) and identified the subclusters of HSCs and GMPs with a distinct molecular signature. RalA also promoted BCR-ABL-induced leukemogenesis and self-renewal of primary LSCs and shortened the survival of leukemic mice. RalA knockdown prolonged survival and promoted sensitivity to imatinib in a patient-derived tumor xenograft model. Immunoprecipitation plus single-cell RNA sequencing of the GMP population confirmed that RalA induced this effect by interacting with RAC1. RAC1 inhibition by azathioprine effectively reduced the self-renewal, colony formation ability of LSCs and prolonged the survival in BCR-ABL1-driven RalA overexpression CML mice. Collectively, RalA was detected to be a vital factor that regulates the abilities of HSCs and LSCs, thus facilitating BCR-ABL-triggered leukemia in mice. RalA inhibition serves as the therapeutic approach to eradicate LSCs in CML.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Fudge JB (2023)

Cardiac defect corrected in vivo with CRISPR.

Nature biotechnology, 41(3):323.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Dyke E, Bijnagte-Schoenmaker C, Wu KM, et al (2023)

Generation of induced pluripotent stem cell line carrying frameshift variants in NPHP1 (UCSFi001-A-68) using CRISPR/Cas9.

Stem cell research, 68:103053.

NPHP1 (Nephrocystin 1) is a protein that localizes to the transition zone of the cilium, a small organelle that projects from the plasma membrane of most cells and allows for integration and coordination of signalling pathways during development and homeostasis. Loss of NPHP1 function due to biallelic NPHP1 gene mutations can lead to the development of ciliopathies - a heterogeneous spectra of disorders characterized by ciliary dysfunction. Here we report the generation of an NPHP1-null hiPSC line (UCSFi001-A-68) via CRISPR/Cas9-mediated non-homologous end joining in the UCSFi001-A background, for study of the role that this protein plays in different tissues.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Zhu Y, J Champer (2023)

Simulations Reveal High Efficiency and Confinement of a Population Suppression CRISPR Toxin-Antidote Gene Drive.

ACS synthetic biology, 12(3):809-819.

Though engineered gene drives hold great promise for spreading through and suppressing populations of disease vectors or invasive species, complications such as resistance alleles and spatial population structure can prevent their success. Additionally, most forms of suppression drives, such as homing drives or driving Y chromosomes, will generally spread uncontrollably between populations with even small levels of migration. The previously proposed CRISPR-based toxin-antidote system called toxin-antidote dominant embryo (TADE) suppression drive could potentially address the issues of confinement and resistance. However, it is a relatively weak form of drive compared to homing drives, which might make it particularly vulnerable to spatial population structure. In this study, we investigate TADE suppression drive using individual-based simulations in a continuous spatial landscape. We find that the drive is actually more confined than in simple models without space, even in its most efficient form with low cleavage rate in embryos from maternally deposited Cas9. Furthermore, the drive performed well in continuous space scenarios if the initial release requirements were met, suppressing the population in a timely manner without being severely affected by chasing, a phenomenon in which wild-type individuals avoid the drive by recolonizing empty areas. At higher embryo cut rates, the drive loses its ability to spread, but a single, widespread release can often still induce rapid population collapse. Thus, if TADE suppression gene drives can be successfully constructed, they may play an important role in control of disease vectors and invasive species when stringent confinement to target populations is desired.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Du L, Zhang D, Luo Z, et al (2023)

Molecular basis of stepwise cyclic tetra-adenylate cleavage by the type III CRISPR ring nuclease Crn1/Sso2081.

Nucleic acids research, 51(5):2485-2495.

The cyclic oligoadenylates (cOAs) act as second messengers of the type III CRISPR immunity system through activating the auxiliary nucleases for indiscriminate RNA degradation. The cOA-degrading nucleases (ring nucleases) provide an 'off-switch' regulation of the signaling, thereby preventing cell dormancy or cell death. Here, we describe the crystal structures of the founding member of CRISPR-associated ring nuclease 1 (Crn1) Sso2081 from Saccharolobus solfataricus, alone, bound to phosphate ions or cA4 in both pre-cleavage and cleavage intermediate states. These structures together with biochemical characterizations establish the molecular basis of cA4 recognition and catalysis by Sso2081. The conformational changes in the C-terminal helical insert upon the binding of phosphate ions or cA4 reveal a gate-locking mechanism for ligand binding. The critical residues and motifs identified in this study provide a new insight to distinguish between cOA-degrading and -nondegrading CARF domain-containing proteins.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Ham O, Kim S, Lee Y, et al (2023)

Generation of telomeric repeat binding factor 1 (TRF1)-knockout human embryonic stem cell lines, KRIBBe010-A-95, KRIBBe010-A-96, and KRIBBe010-A-97, using CRISPR/Cas9 technology.

Stem cell research, 68:103045.

Telomeric repeat binding factor 1 (TRF1) plays an essential role in maintaining telomere length. Here, we established TRF1-knockout human pluripotent stem cells (hPSCs; hTRF1-KO) using the CRISPR/Cas9 technology. The hTRF1-KO cell lines expressed pluripotency markers and demonstrated a normal karyotype (46, XX) and DNA profile. In addition, hTRF1-KOcells spontaneously differentiated into all three germ layers in vitro. Thus, these cell lines could be useful models in various research fields.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Huan YW, Torraca V, Brown R, et al (2023)

P1 Bacteriophage-Enabled Delivery of CRISPR-Cas9 Antimicrobial Activity Against Shigella flexneri.

ACS synthetic biology, 12(3):709-721.

The discovery of clustered, regularly interspaced, short palindromic repeats (CRISPR) and the Cas9 RNA-guided nuclease provides unprecedented opportunities to selectively kill specific populations or species of bacteria. However, the use of CRISPR-Cas9 to clear bacterial infections in vivo is hampered by the inefficient delivery of cas9 genetic constructs into bacterial cells. Here, we use a broad-host-range P1-derived phagemid to deliver the CRISPR-Cas9 chromosomal-targeting system into Escherichia coli and the dysentery-causing Shigella flexneri to achieve DNA sequence-specific killing of targeted bacterial cells. We show that genetic modification of the helper P1 phage DNA packaging site (pac) significantly enhances the purity of packaged phagemid and improves the Cas9-mediated killing of S. flexneri cells. We further demonstrate that P1 phage particles can deliver chromosomal-targeting cas9 phagemids into S. flexneri in vivo using a zebrafish larvae infection model, where they significantly reduce the bacterial load and promote host survival. Our study highlights the potential of combining P1 bacteriophage-based delivery with the CRISPR chromosomal-targeting system to achieve DNA sequence-specific cell lethality and efficient clearance of bacterial infection.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Stępniewski J, Jeż M, J Dulak (2023)

Generation of miR-15a/16-1 cluster-deficient human induced pluripotent stem cell line (DMBi001-A-2) using CRISPR/Cas9 gene editing.

Stem cell research, 68:103046.

miR-15a/16-1 cluster, composed of MIR15A and MIR16-1 genes located in close proximity on chromosome 13 was described to regulate post-natal cell cycle withdrawal of cardiomyocytes in mice. In humans, on the other hand, the level of miR-15a-5p and miR-16-p was negatively associated with the severity of cardiac hypertrophy. Therefore, to better understand the role of these microRNAs in human cardiomyocytes in regard to their proliferative potential and hypertrophic growth, we generated hiPSC line with complete deletion of miR-15a/16-1 cluster using CRISPR/Cas9 gene editing. Obtained cells demonstrate expression of pluripotency markers, differentiation capacity into all three germ layers and normal karyotype.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Jeong SH, Kim HJ, SJ Lee (2023)

New Target Gene Screening Using Shortened and Random sgRNA Libraries in Microbial CRISPR Interference.

ACS synthetic biology, 12(3):800-808.

CRISPR interference (CRISPRi) screening has been used for identification of target genes related to specific phenotypes using single-molecular guide RNA (sgRNA) libraries. In CRISPRi screening, the sizes of random sgRNA libraries contained with the original target recognition sequences are large (∼10[12]). Here, we demonstrated that the length of the target recognition sequence (TRS) can be shortened in sgRNAs from the original 20 nucleotides (N20) to 9 nucleotides (N9) that is still sufficient for dCas9 to repress target genes in the xylose operon of Escherichia coli, regardless of binding to a promoter or open reading frame region. Based on the results, we constructed random sgRNA plasmid libraries with 5'-shortened TRS lengths, and identified xylose metabolic target genes by Sanger sequencing of sgRNA plasmids purified from Xyl[-] phenotypic cells. Next, the random sgRNA libraries were harnessed to screen for target genes to enhance violacein pigment production in synthetic E. coli cells. Seventeen target genes were selected by analyzing the redundancy of the TRS in sgRNA plasmids in dark purple colonies. Among them, seven genes (tyrR, pykF, cra, ptsG, pykA, sdaA, and tnaA) have been known to increase the intracellular l-tryptophan pool, the precursor of a violacein. Seventeen cells with a single deletion of each target gene exhibited a significant increase in violacein production. These results indicate that using shortened random TRS libraries for CRISPRi can be simple and cost-effective for phenotype-based target gene screening.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Nguyen TTT, Tanaka Y, Sanada M, et al (2023)

CRISPR/Cas9-Mediated Induction of Relapse-Specific NT5C2 and PRPS1 Mutations Confers Thiopurine Resistance as a Relapsed Lymphoid Leukemia Model.

Molecular pharmacology, 103(4):199-210.

6-Mercaptopurine (6-MP) is a key component in maintenance therapy for childhood acute lymphoblastic leukemia (ALL). Recent next-generation sequencing analysis of childhood ALL clarified the emergence of the relapse-specific mutations of the NT5C2 and PRPS1 genes, which are involved in thiopurine metabolism. In this scenario, minor clones of leukemia cells could acquire the 6-MP-resistant phenotype as a result of the NT5C2 or PRPS1 mutation during chemotherapy (including 6-MP treatment) and confer disease relapse after selective expansion. Thus, to establish new therapeutic modalities overcoming 6-MP resistance in relapsed ALL, human leukemia models with NT5C2 and PRPS1 mutations in the intrinsic genes are urgently required. Here, mimicking the initiation process of the above clinical course, we sought to induce two relapse-specific hotspot mutations (R39Q mutation of the NT5C2 gene and S103N mutation of the PRPS1 gene) into a human lymphoid leukemia cell line by homologous recombination (HR) using the CRISPR/Cas9 system. After 6-MP selection of the cells transfected with Cas9 combined with single-guide RNA and donor DNA templates specific for either of those two mutations, we obtained the sublines with the intended NT5C2-R39Q and PRPS1-S103N mutation as a result of HR. Moreover, diverse in-frame small insertion/deletions were also confirmed in the 6-MP-resistant sublines at the target sites of the NT5C2 and PRPS1 genes as a result of nonhomologous end joining. These sublines are useful for molecular pharmacological evaluation of the NT5C2 and PRPS1 gene mutations in the 6-MP sensitivity and development of therapy overcoming the thiopurine resistance of leukemia cells. SIGNIFICANCE STATEMENT: Mimicking the initiation process of relapse-specific mutations of the NT5C2 and PRPS1 genes in childhood acute lymphoblastic leukemia treated with 6-mercaptopurine (6-MP), this study sought to introduce NT5C2-R39Q and PRPS1-S103N mutations into a human lymphoid leukemia cell line by homologous recombination using the CRISPR/Cas9 system. In the resultant 6-MP-resistant sublines, the intended mutations and diverse in-frame small insertions/deletions were confirmed, indicating that the obtained sublines are useful for molecular pharmacological evaluation of the NT5C2 and PRPS1 gene mutations.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Lackner M, Helmbrecht N, Pääbo S, et al (2023)

Detection of unintended on-target effects in CRISPR genome editing by DNA donors carrying diagnostic substitutions.

Nucleic acids research, 51(5):e26.

CRISPR nucleases can introduce double-stranded DNA breaks in genomes at positions specified by guide RNAs. When repaired by the cell, this may result in the introduction of insertions and deletions or nucleotide substitutions provided by exogenous DNA donors. However, cellular repair can also result in unintended on-target effects, primarily larger deletions and loss of heterozygosity due to gene conversion. Here we present a strategy that allows easy and reliable detection of unintended on-target effects as well as the generation of control cells that carry wild-type alleles but have demonstratively undergone genome editing at the target site. Our 'sequence-ascertained favorable editing' (SAFE) donor approach relies on the use of DNA donor mixtures containing the desired nucleotide substitutions or the wild-type alleles together with combinations of additional 'diagnostic' substitutions unlikely to have any effects. Sequencing of the target sites then results in that two different sequences are seen when both chromosomes are edited with 'SAFE' donors containing different sets of substitutions, while a single sequence indicates unintended effects such as deletions or gene conversion. We analyzed more than 850 human embryonic stem cell clones edited with 'SAFE' donors and detect all copy number changes and almost all clones with gene conversion.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Zulhussnain M, Zahoor MK, Ranian K, et al (2023)

CRISPR Cas9 mediated knockout of sex determination pathway genes in Aedes aegypti.

Bulletin of entomological research, 113(2):243-252.

The vector role of Aedes aegypti for viral diseases including dengue and dengue hemorrhagic fever makes it imperative for its proper control. Despite various adopted control strategies, genetic control measures have been recently focused against this vector. CRISPR Cas9 system is a recent and most efficient gene editing tool to target the sex determination pathway genes in Ae. aegypti. In the present study, CRISPR Cas9 system was used to knockout Ae. aegypti doublesex (Aaedsx) and Ae. aegypti sexlethal (AaeSxl) genes in Ae. aegypti embryos. The injection mixes with Cas9 protein (333 ng ul[-1]) and gRNAs (each at 100 ng ul[-1]) were injected into eggs. Injected eggs were allowed to hatch at 26 ± 1°C, 60 ± 10% RH. The survival and mortality rate was recorded in knockout Aaedsx and AaeSxl. The results revealed that knockout produced low survival and high mortality. A significant percentage of eggs (38.33%) did not hatch as compared to control groups (P value 0.00). Highest larval mortality (11.66%) was found in the knockout of Aaedsx female isoform, whereas, the emergence of only male adults also showed that the knockout of Aaedsx (female isoform) does not produce male lethality. The survival (3.33%) of knockout for AaeSxl eggs to the normal adults suggested further study to investigate AaeSxl as an efficient upstream of Aaedsx to target for sex transformation in Ae. aegypti mosquitoes.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Lee S, Lee H, Baek G, et al (2023)

Precision mitochondrial DNA editing with high-fidelity DddA-derived base editors.

Nature biotechnology, 41(3):378-386.

Bacterial toxin DddA-derived cytosine base editors (DdCBEs)-composed of split DddAtox (a cytosine deaminase specific to double-stranded DNA), custom-designed TALE (transcription activator-like effector) DNA-binding proteins, and a uracil glycosylase inhibitor-enable mitochondrial DNA (mtDNA) editing in human cells, which may pave the way for therapeutic correction of pathogenic mtDNA mutations in patients. The utility of DdCBEs has been limited by off-target activity, which is probably caused by spontaneous assembly of the split DddAtox deaminase enzyme, independent of DNA-binding interactions. We engineered high-fidelity DddA-derived cytosine base editors (HiFi-DdCBEs) with minimal off-target activity by substituting alanine for amino acid residues at the interface between the split DddAtox halves. The resulting domains cannot form a functional deaminase without binding of their linked TALE proteins at adjacent sites on DNA. Whole mitochondrial genome sequencing shows that, unlike conventional DdCBEs, which induce hundreds of unwanted off-target C-to-T conversions in human mtDNA, HiFi-DdCBEs are highly efficient and precise, avoiding collateral off-target mutations, and as such, they will probably be desirable for therapeutic applications.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Christie KA, Guo JA, Silverstein RA, et al (2023)

Precise DNA cleavage using CRISPR-SpRYgests.

Nature biotechnology, 41(3):409-416.

Methods for in vitro DNA cleavage and molecular cloning remain unable to precisely cleave DNA directly adjacent to bases of interest. Restriction enzymes (REs) must bind specific motifs, whereas wild-type CRISPR-Cas9 or CRISPR-Cas12 nucleases require protospacer adjacent motifs (PAMs). Here we explore the utility of our previously reported near-PAMless SpCas9 variant, named SpRY, to serve as a universal DNA cleavage tool for various cloning applications. By performing SpRY DNA digests (SpRYgests) using more than 130 guide RNAs (gRNAs) sampling a wide diversity of PAMs, we discovered that SpRY is PAMless in vitro and can cleave DNA at practically any sequence, including sites refractory to cleavage with wild-type SpCas9. We illustrate the versatility and effectiveness of SpRYgests to improve the precision of several cloning workflows, including those not possible with REs or canonical CRISPR nucleases. We also optimize a rapid and simple one-pot gRNA synthesis protocol to streamline SpRYgest implementation. Together, SpRYgests can improve various DNA engineering applications that benefit from precise DNA breaks.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Grünewald J, Miller BR, Szalay RN, et al (2023)

Engineered CRISPR prime editors with compact, untethered reverse transcriptases.

Nature biotechnology, 41(3):337-343.

The CRISPR prime editor PE2 consists of a Streptococcus pyogenes Cas9 nickase (nSpCas9) fused at its C-terminus to a Moloney murine leukemia virus reverse transcriptase (MMLV-RT). Here we show that separated nSpCas9 and MMLV-RT proteins function as efficiently as intact PE2 in human cells. We use this Split-PE system to rapidly identify and engineer more compact prime editor architectures that also broaden the types of RTs used for prime editing.

RevDate: 2023-03-18

Zakrzewska M, M Burmistrz (2023)

Mechanisms regulating the CRISPR-Cas systems.

Frontiers in microbiology, 14:1060337.

The CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats- CRISPR associated proteins) is a prokaryotic system that enables sequence specific recognition and cleavage of nucleic acids. This is possible due to cooperation between CRISPR array which contains short fragments of DNA called spacers that are complimentary to the targeted nucleic acid and Cas proteins, which take part in processes of: acquisition of new spacers, processing them into their functional form as well as recognition and cleavage of targeted nucleic acids. The primary role of CRISPR-Cas systems is to provide their host with an adaptive and hereditary immunity against exogenous nucleic acids. This system is present in many variants in both Bacteria and Archea. Due to its modular structure, and programmability CRISPR-Cas system become attractive tool for modern molecular biology. Since their discovery and implementation, the CRISPR-Cas systems revolutionized areas of gene editing and regulation of gene expression. Although our knowledge on how CRISPR-Cas systems work has increased rapidly in recent years, there is still little information on how these systems are controlled and how they interact with other cellular mechanisms. Such regulation can be the result of both auto-regulatory mechanisms as well as exogenous proteins of phage origin. Better understanding of these interaction networks would be beneficial for optimization of current and development of new CRISPR-Cas-based tools. In this review we summarize current knowledge on the various molecular mechanisms that affect activity of CRISPR-Cas systems.

RevDate: 2023-03-18

Cerbino GN, Traglia GM, Ayala Nuñez T, et al (2023)

Comparative genome analysis of the genus Shewanella unravels the association of key genetic traits with known and potential pathogenic lineages.

Frontiers in microbiology, 14:1124225.

Shewanella spp. are Gram-negative rods widely disseminated in aquatic niches that can also be found in human-associated environments. In recent years, reports of infections caused by these bacteria have increased significantly. Mobilome and resistome analysis of a few species showed that they are versatile; however, comprehensive comparative studies in the genus are lacking. Here, we analyzed the genetic traits of 144 genomes from Shewanella spp. isolates focusing on the mobilome, resistome, and virulome to establish their evolutionary relationship and detect unique features based on their genome content and habitat. Shewanella spp. showed a great diversity of mobile genetic elements (MGEs), most of them associated with monophyletic lineages of clinical isolates. Furthermore, 79/144 genomes encoded at least one antimicrobial resistant gene with their highest occurrence in clinical-related lineages. CRISPR-Cas systems, which confer immunity against MGEs, were found in 41 genomes being I-E and I-F the more frequent ones. Virulome analysis showed that all Shewanella spp. encoded different virulence genes (motility, quorum sensing, biofilm, adherence, etc.) that may confer adaptive advantages for survival against hosts. Our data revealed that key accessory genes are frequently found in two major clinical-related groups, which encompass the opportunistic pathogens Shewanella algae and Shewanella xiamenensis together with several other species. This work highlights the evolutionary nature of Shewanella spp. genomes, capable of acquiring different key genetic traits that contribute to their adaptation to different niches and facilitate the emergence of more resistant and virulent isolates that impact directly on human and animal health.

RevDate: 2023-03-18

Pedrazzoli E, Bianchi A, Umbach A, et al (2023)

An optimized SpCas9 high-fidelity variant for direct protein delivery.

Molecular therapy : the journal of the American Society of Gene Therapy pii:S1525-0016(23)00128-4 [Epub ahead of print].

Electroporation of the Cas9 ribonucleoprotein (RNP) complex offers the advantage of preventing off-target cleavages and potential immune responses produced by long-term expression of the nuclease. Nevertheless, the majority of engineered high-fidelity Streptococcus pyogenes Cas9 (SpCas9) variants are less active than the wild-type enzyme and are not compatible with RNP delivery. Building on our previous studies on evoCas9, we developed a high-fidelity SpCas9 variant suitable for RNP delivery. The editing efficacy and precision of the recombinant high-fidelity Cas9 (rCas9HF), characterized by the K526D substitution, was compared with the R691A mutant (HiFi Cas9), which is currently the only available high-fidelity Cas9 that can be used as an RNP. The comparative analysis was extended to gene substitution experiments where the two high fidelities were used in combination with a DNA donor template, generating different ratios of non-homologous end joining (NHEJ) versus homology-directed repair (HDR) for precise editing. The analyses revealed a heterogeneous efficacy and precision indicating different targeting capabilities between the two variants throughout the genome. The development of rCas9HF, characterized by an editing profile diverse from the currently used HiFi Cas9 in RNP electroporation, increases the genome editing solutions for the highest precision and efficient applications.

RevDate: 2023-03-16

Wang X, Mu X, Li J, et al (2023)

A novel nanoparticle surface-constrained CRISPR-Cas12a 3D DNA walker-like nanomachines for sensitive and stable miRNAs detection.

Analytica chimica acta, 1251:340950.

The CRISPR-Cas system has broad prospects as a new type of nucleic acid signal amplification technology based on the trans-cleavage activity of Cas12a to single-stranded DNA, but the trans-cleavage reaction efficiency is relatively low in solution. In order to overcome this negative factor, a new 3D DNA nanomachine whose CRISPR-Cas12a is limited to the surface of nanoparticles is used for sensitive and stable detection of miRNA. By loading Cas12a activator onto spherical nucleic acid (SNA), the CRISPR-Cas12a activator system on the surface of Au nanoparticles (AuNPs) acts as a walker to carry out continuous recognition-walking-cutting reaction on the surface of AuNPs, which enhances the trans-cleavage activity of Cas12a to SNAs. Benefiting from the confinement effect of spherical nucleic acids surface, a 3D DNA nanomachine has been developed for the detection of miRNA-21, which has achieved high sensitivity and accuracy, and the detection limit is able to reach 8.0 pM. This new 3D DNA walker-like nanomachine provided another insight for future bioanalysis and early clinical diagnoses of disease and liquid biopsy.

RevDate: 2023-03-16

Mikkelsen NS, Hernandez SS, Jensen TI, et al (2023)

Enrichment of transgene integrations by transient CRISPR activation of a silent reporter gene.

Molecular therapy. Methods & clinical development, 29:1-16.

CRISPR-Cas-mediated site-specific integration of transgenes by homology-directed repair (HDR) is challenging, especially in primary cells, where inferior editing efficiency may impede the development of gene- and cellular therapies. Various strategies for enrichment of cells with transgene integrations have been developed, but most strategies either generate unwanted genomic scars or rely on permanent integration and expression of a reporter gene used for selection. However, stable expression of a reporter gene may perturb cell homeostasis and function. Here we develop a broadly applicable and versatile enrichment strategy by harnessing the capability of CRISPR activation (CRISPRa) to transiently induce expression of a therapeutically relevant reporter gene used for immunomagnetic enrichment. This strategy is readily adaptable to primary human T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs), where enrichment of 1.8- to 3.3-fold and 3.2- to 3.6-fold was achieved, respectively. Furthermore, chimeric antigen receptor (CAR) T cells were enriched 2.5-fold and demonstrated improved cytotoxicity over non-enriched CAR T cells. Analysis of HDR integrations showed a proportion of cells harboring deletions of the transgene cassette arising either from impartial HDR or truncated adeno-associated virus (AAV) vector genomes. Nonetheless, this novel enrichment strategy expands the possibility to enrich for transgene integrations in research settings and in gene and cellular therapies.

RevDate: 2023-03-17
CmpDate: 2023-03-16

Jianwei L, Jobichen C, Machida S, et al (2023)

Structures of apo Cas12a and its complex with crRNA and DNA reveal the dynamics of ternary complex formation and target DNA cleavage.

PLoS biology, 21(3):e3002023.

Cas12a is a programmable nuclease for adaptive immunity against invading nucleic acids in CRISPR-Cas systems. Here, we report the crystal structures of apo Cas12a from Lachnospiraceae bacterium MA2020 (Lb2) and the Lb2Cas12a+crRNA complex, as well as the cryo-EM structure and functional studies of the Lb2Cas12a+crRNA+DNA complex. We demonstrate that apo Lb2Cas12a assumes a unique, elongated conformation, whereas the Lb2Cas12a+crRNA binary complex exhibits a compact conformation that subsequently rearranges to a semi-open conformation in the Lb2Cas12a+crRNA+DNA ternary complex. Notably, in solution, apo Lb2Cas12a is dynamic and can exist in both elongated and compact forms. Residues from Met493 to Leu523 of the WED domain undergo major conformational changes to facilitate the required structural rearrangements. The REC lobe of Lb2Cas12a rotates 103° concomitant with rearrangement of the hinge region close to the WED and RuvC II domains to position the RNA-DNA duplex near the catalytic site. Our findings provide insight into crRNA recognition and the mechanism of target DNA cleavage.

RevDate: 2023-03-14

Spasskaya DS, Davletshin AI, Bachurin SS, et al (2023)

Improving the on-target activity of high-fidelity Cas9 editors by combining rational design and random mutagenesis.

Applied microbiology and biotechnology [Epub ahead of print].

Genomic and post-genomic editors based on CRISPR/Cas systems are widely used in basic research and applied sciences, including human gene therapy. Most genome editing tools are based on the CRISPR/Cas9 type IIA system from Streptococcus pyogenes. Unfortunately, a number of drawbacks have hindered its application in therapeutic approaches, the most serious of which is the relatively high level of off-targets. To overcome this obstacle, various high-fidelity Cas9 variants have been created. However, they show reduced on-target activity compared to wild-type Cas9 possibly due to increased sensitivity to eukaryotic chromatin. Here, we combined a rational approach with random mutagenesis to create a set of new Cas9 variants showing high specificity and increased activity in Saccharomyces cerevisiae yeast. Moreover, a novel mutation in the PAM (protospacer adjacent motif)-interacting Cas9 domain was found, which increases the on-target activity of high-fidelity Cas9 variants while retaining their high specificity. The obtained data suggest that this mutation acts by weakening the eukaryotic chromatin barrier for Cas9 and rearranging the RuvC active center. Improved Cas9 variants should further advance genome and post-genome editing technologies. KEY POINTS: • D147Y and P411T mutations increase the activity of high-fidelity Cas9 variants. • The new L1206P mutation further increases the activity of high-fidelity Cas9 variants. • The L1206P mutation weakens the chromatin barrier for Cas9 editors.

RevDate: 2023-03-15
CmpDate: 2023-03-15

Wang D, Fletcher GC, Gagic D, et al (2023)

Comparative genome identification of accessory genes associated with strong biofilm formation in Vibrio parahaemolyticus.

Food research international (Ottawa, Ont.), 166:112605.

Vibrio parahaemolyticus biofilms on the seafood processing plant surfaces are a potential source of seafood contamination and subsequent food poisoning. Strains differ in their ability to form biofilm, but little is known about the genetic characteristics responsible for biofilm development. In this study, pangenome and comparative genome analysis of V. parahaemolyticus strains reveals genetic attributes and gene repertoire that contribute to robust biofilm formation. The study identified 136 accessory genes that were exclusively present in strong biofilm forming strains and these were functionally assigned to the Gene Ontology (GO) pathways of cellulose biosynthesis, rhamnose metabolic and catabolic processes, UDP-glucose processes and O antigen biosynthesis (p < 0.05). Strategies of CRISPR-Cas defence and MSHA pilus-led attachment were implicated via Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation. Higher levels of horizontal gene transfer (HGT) were inferred to confer more putatively novel properties on biofilm-forming V. parahaemolyticus. Furthermore, cellulose biosynthesis, a neglected potential virulence factor, was identified as being acquired from within the order Vibrionales. The cellulose synthase operons in V. parahaemolyticus were examined for their prevalence (22/138, 15.94 %) and were found to consist of the genes bcsG, bcsE, bcsQ, bcsA, bcsB, bcsZ, bcsC. This study provides insights into robust biofilm formation of V. parahaemolyticus at the genomic level and facilitates: identification of key attributes for robust biofilm formation, elucidation of biofilm formation mechanisms and development of potential targets for novel control strategies of persistent V. parahaemolyticus.

RevDate: 2023-03-15
CmpDate: 2023-03-15

Adeyinka OS, Tabassum B, Koloko BL, et al (2023)

Enhancing the quality of staple food crops through CRISPR/Cas-mediated site-directed mutagenesis.

Planta, 257(4):78.

The enhancement of CRISPR-Cas gene editing with robust nuclease activity promotes genetic modification of desirable agronomic traits, such as resistance to pathogens, drought tolerance, nutritional value, and yield-related traits in crops. The genetic diversity of food crops has reduced tremendously over the past twelve millennia due to plant domestication. This reduction presents significant challenges for the future especially considering the risks posed by global climate change to food production. While crops with improved phenotypes have been generated through crossbreeding, mutation breeding, and transgenic breeding over the years, improving phenotypic traits through precise genetic diversification has been challenging. The challenges are broadly associated with the randomness of genetic recombination and conventional mutagenesis. This review highlights how emerging gene-editing technologies reduce the burden and time necessary for developing desired traits in plants. Our focus is to provide readers with an overview of the advances in CRISPR-Cas-based genome editing for crop improvement. The use of CRISPR-Cas systems in generating genetic diversity to enhance the quality and nutritional value of staple food crops is discussed. We also outlined recent applications of CRISPR-Cas in developing pest-resistant crops and removing unwanted traits, such as allergenicity from crops. Genome editing tools continue to evolve and present unprecedented opportunities to enhance crop germplasm via precise mutations at the desired loci of the plant genome.

RevDate: 2023-03-16
CmpDate: 2023-03-16

Chen J, Shi G, C Yan (2023)

Portable biosensor for on-site detection of kanamycin in water samples based on CRISPR-Cas12a and an off-the-shelf glucometer.

The Science of the total environment, 872:162279.

On-site and cost-effective monitoring of antibiotic residue in water samples using a ubiquitous device that is readily available to the general public is a big challenge. Herein, we developed a portable biosensor for kanamycin (KAN) detection based on a glucometer and CRISPR-Cas12a. The aptamer-KAN interactions liberate the trigger C strand, which can initiate the hairpin assembly to produce numerous double-stranded DNA. After recognition by CRISPR-Cas12a, Cas12a can cleave the magnetic bead and invertase-modified single-stranded DNA. After magnetic separation, the invertase can convert sucrose into glucose, which can be quantified by a glucometer. The linear range of the glucometer biosensor is from 1 pM to 100 nM and the detection limit is 1 pM. The biosensor also exhibited high selectivity and the nontarget antibiotics had no significant interference with KAN detection. The sensing system is robust and can work in complex samples with excellent accuracy and reliability. The recovery values were in the range of 89-107.2 % for water samples and 86-106.5 % for milk samples. The relative standard deviation (RSD) was below 5 %. With the advantages of simple operation, low cost, and easy accessibility to the public, this portable pocket-sized sensor can realize the on-site detection of antibiotic residue in resource-limited settings.

RevDate: 2023-03-16
CmpDate: 2023-03-16

Abhinandan K, Hickerson NMN, Lan X, et al (2023)

Disabling of ARC1 through CRISPR-Cas9 leads to a complete breakdown of self-incompatibility responses in Brassica napus.

Plant communications, 4(2):100504.

RevDate: 2023-03-15
CmpDate: 2023-03-15

Ma R, Jiang J, Ya Y, et al (2023)

A carbon dot-based nanoscale covalent organic framework as a new emitter combined with a CRISPR/Cas12a-mediated electrochemiluminescence biosensor for ultrasensitive detection of bisphenol A.

The Analyst, 148(6):1362-1370.

Exploring new highly efficient electrochemiluminescence (ECL) luminophores is a necessary condition for developing ultrasensitive ECL biosensors. Therefore, a luminescent carbon dot-based covalent organic framework (CD-COF) was prepared using aldehyde-based carbon dots (CDs) and 1,3,5-tris (4-aminophenyl) benzene (TPB). Because the CD-COF made the regular arrangement of CDs conducive to improving the ECL response, CD-COF had a higher ECL intensity and efficiency than CDs. What's more, the ECL intensity of the CD-COF/S2O8[2-]/Bu4N[+] system was about 2.98, 7.50, and 28.08 times higher than those of the CD-COF/S2O8[2-], CDs/S2O8[2-] and S2O8[2-] systems, respectively. Considering the remarkable ECL performance, the CD-COF/S2O8[2-]/Bu4N[+] system was employed combined with the CRISPR/Cas12a trans-cutting strategy to construct an "off-on" ECL biosensor for BPA detection. The proposed ECL biosensor exhibited excellent performance with a wide linear range from 1.0 × 10[-14] mol L[-1] to 1.0 × 10[-5] mol L[-1] with a low detection limit of 2.21 fM (S/N = 3) under the optimized conditions. The biosensor demonstrated that CD-COF can be used as an efficient ECL emitter, thus expanding the application field of COFs. In addition, the good stability and specificity of the biosensor enabled the rapid detection of BPA, which will provide valuable insights into promising ultrasensitive ECL biosensors.

RevDate: 2023-03-15
CmpDate: 2023-03-15

Puppulin L, Ishikawa J, Sumino A, et al (2023)

Dynamics of Target DNA Binding and Cleavage by Staphylococcus aureus Cas9 as Revealed by High-Speed Atomic Force Microscopy.

ACS nano, 17(5):4629-4641.

Programmable DNA binding and cleavage by CRISPR-Cas9 has revolutionized the life sciences. However, the off-target cleavage observed in DNA sequences with some homology to the target still represents a major limitation for a more widespread use of Cas9 in biology and medicine. For this reason, complete understanding of the dynamics of DNA binding, interrogation and cleavage by Cas9 is crucial to improve the efficiency of genome editing. Here, we use high-speed atomic force microscopy (HS-AFM) to investigate Staphylococcus aureus Cas9 (SaCas9) and its dynamics of DNA binding and cleavage. Upon binding to single-guide RNA (sgRNA), SaCas9 forms a close bilobed structure that transiently and flexibly adopts also an open configuration. The SaCas9-mediated DNA cleavage is characterized by release of cleaved DNA and immediate dissociation, confirming that SaCas9 operates as a multiple turnover endonuclease. According to present knowledge, the process of searching for target DNA is mainly governed by three-dimensional diffusion. Independent HS-AFM experiments show a potential long-range attractive interaction between SaCas9-sgRNA and its target DNA. The interaction precedes the formation of the stable ternary complex and is observed exclusively in the vicinity of the protospacer-adjacent motif (PAM), up to distances of several nanometers. The direct visualization of the process by sequential topographic images suggests that SaCas9-sgRNA binds to the target sequence first, while the following binding of the PAM is accompanied by local DNA bending and formation of the stable complex. Collectively, our HS-AFM data reveal a potential and unexpected behavior of SaCas9 during the search for DNA targets.

RevDate: 2023-03-15
CmpDate: 2023-03-15

Senthilnathan R, Ilangovan I, Kunale M, et al (2023)

An update on CRISPR-Cas12 as a versatile tool in genome editing.

Molecular biology reports, 50(3):2865-2881.

Gene editing techniques, which help in modification of any DNA sequence at ease, have revolutionized the world of Genetic engineering. Although there are other gene-editing techniques, CRISPR has emerged as the chief and most preferred tool due to its simplicity and capacity to execute effective gene editing in a wide range of organisms. Although Cas9 has widely been employed for genetic modification over the years, Cas12 systems have lately emerged as a viable option. This review primarily focuses on assessing Cas12-mediated mutagenesis and elucidating the editing efficacy of both Cpf1 (Cas12a) and C2c1 (Cas12b) systems in microbes, plants, and other species. Also, we reviewed several genetic alterations that have been performed with these Cas12 systems to improve editing efficiency. Furthermore, the experimental benefits and applications of Cas12 systems are highlighted in this study.

RevDate: 2023-03-15
CmpDate: 2023-03-15

Han JY, Seo J, Choi Y, et al (2023)

CRISPR-Cas9 mediated genome editing of Huntington's disease neurospheres.

Molecular biology reports, 50(3):2127-2136.

BACKGROUND: Huntington's disease (HD) is a fatal genetic disease caused by polyglutamine aggregation encoded by an expanded CAG repeat in the huntingtin gene (HTT). In this study, we cultured neurospheres derived from R6/2 mice, a representative animal model of HD, as an in vitro model. GuideRNAs were designed to induce large deletion or frameshift indel mutation of CAG expansion. These gRNAs and Cas9 were delivered to the R6/2 neurospheres and disease-related phenotypes were observed.

METHODS AND RESULTS: Deletion or indel mutation of the CAG repeat was confirmed by PCR, T7E1 assay and sequencing of the edited neurospheres. Edited neurospheres showed decreased polyglutamine aggregation compared with control HD neurospheres. In the edited neurosphere, we confirmed the upregulation of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) and brain-derived neurotrophic factor (BDNF), whose reduced expressions are closely involved in the disease progression. In addition, flow cytometry result showed an increase in cell viability with an overall decrease in necrotic and apoptotic populations among edited R6/2 neurospheres. Additional siRNA experiments confirmed that the increased viability was decreased through inhibition of PGC-1α or BDNF.

CONCLUSION: Our study confirmed that CAG repeat of R6/2 mouse-derived neurospheres can be edited through CRISPR-Cas9. Editing of CAG repeat sequence decreases polyglutamine aggregation and cellular apoptosis of HD neurospheres, which may be related to the increased expressions of PGC-1α and BDNF. Our data provide the evidence that CRISPR-Cas9 mediated genome editing has therapeutic potential on HD neuronal cells.

RevDate: 2023-03-15
CmpDate: 2023-03-15

Zhang Y, Blahut-Beatty L, Zheng S, et al (2023)

The Role of a Soybean 14-3-3 Gene (Glyma05g29080) on White Mold Resistance and Nodulation Investigations Using CRISPR-Cas9 Editing and RNA Silencing.

Molecular plant-microbe interactions : MPMI, 36(3):159-164.

The role of a soybean 14-3-3 gene (Glyma05g29080) in defense against white mold and in nodulation was investigated by loss-of-gene-function with CRISPR-Cas9 editing and silencing of RNA interference (RNAi). Particle bombardment was used to introduce the CRISPR expression cassette to target the soybean 14-3-3 gene and an RNAi construct to silence gene transcription. Transmission of the edited 14-3-3 gene and the RNAi construct was confirmed in their respective progeny. The recovered transgenic plants and their progeny were significantly more susceptible to Sclerotinia sclerotiorum infection and showed a significant reduction in nodulation, thus confirming the role of the 14-3-3 gene (Glyma05g29080) in both nodulation and defense.

RevDate: 2023-03-13

Timberlake AT (2023)

Molecular scalpels: the future of pediatric craniofacial surgery?.

Plastic and reconstructive surgery pii:00006534-990000000-01651 [Epub ahead of print].

CRISPR-Cas genome editing tools are among the most substantial advances in the life sciences in modern history. Single dose gene therapies to correct pathogenic mutations have moved quickly from bench to bedside, with several therapeutics designed by CRISPR pioneers entering various stages of clinical investigation. Applications of these genetic technologies are poised to reshape the practice of both medicine and surgery. Many of the most morbid conditions treated by craniofacial surgeons are syndromic craniosynostoses caused by mutations in fibroblast growth factor receptor (FGFR) genes, including Apert, Pfeiffer, Crouzon, and Muenke syndromes. The fact that pathogenic mutations in these genes are recurrent in the majority of affected families presents a unique opportunity to develop "off the shelf" gene editing therapies to correct these mutations in affected children. The therapeutic potential of these interventions could reshape pediatric craniofacial surgery, potentially first eliminating the need for midface advancement procedures in affected children.

RevDate: 2023-03-13

Wiegand T, Wilkinson R, Santiago-Frangos A, et al (2023)

Functional and Phylogenetic Diversity of Cas10 Proteins.

The CRISPR journal [Epub ahead of print].

Cas10 proteins are large subunits of type III CRISPR RNA (crRNA)-guided surveillance complexes, many of which have nuclease and cyclase activities. Here, we use computational and phylogenetic methods to identify and analyze 2014 Cas10 sequences from genomic and metagenomic databases. Cas10 proteins cluster into five distinct clades that mirror previously established CRISPR-Cas subtypes. Most Cas10 proteins (85.0%) have conserved polymerase active-site motifs, while HD-nuclease domains are less well conserved (36.0%). We identify Cas10 variants that are split over multiple genes or genetically fused to nucleases activated by cyclic nucleotides (i.e., NucC) or components of toxin-antitoxin systems (i.e., AbiEii). To clarify the functional diversification of Cas10 proteins, we cloned, expressed, and purified five representatives from three phylogenetically distinct clades. None of the Cas10s are functional cyclases in isolation, and activity assays performed with polymerase domain active site mutants indicate that previously reported Cas10 DNA-polymerase activity may be a result of contamination. Collectively, this work helps clarify the phylogenetic and functional diversity of Cas10 proteins in type III CRISPR systems.

RevDate: 2023-03-14

Jang G, Shin HR, Do HS, et al (2023)

Therapeutic gene correction for Lesch-Nyhan syndrome using CRISPR-mediated base and prime editing.

Molecular therapy. Nucleic acids, 31:586-595.

Lesch-Nyhan syndrome (LNS) is inherited as an X-linked recessive genetic disorder caused by mutations in hypoxanthine-guanine phosphoribosyl transferase 1 (HPRT1). Patients with LNS show various clinical phenotypes, including hyperuricemia, gout, devastating behavioral abnormality, intellectual disability, and self-harm. Although uric acid overproduction can be modulated with the xanthine oxidase inhibitor allopurinol, there exists no treatment for behavioral and neurological manifestations of LNS. In the current study, CRISPR-mediated base editors (BEs) and prime editors (PEs) were utilized to generate LNS-associated disease models and correct the disease models for therapeutic approach. Cytosine BEs (CBEs) were used to induce c.430C>T and c.508C>T mutations in HAP1 cells, and then adenine BEs (ABEs) were used to correct these mutations without DNA cleavage. PEs induced a c.333_334ins(A) mutation, identified in a Korean patient with LNS, in HAP1 cells, which was corrected in turn by PEs. Furthermore, improved PEs corrected the same mutation in LNS patient-derived fibroblasts by up to 14% without any unwanted mutations. These results suggest that CRISPR-mediated BEs and PEs would be suggested as a potential therapeutic strategy of this extremely rare, devastating genetic disease.

RevDate: 2023-03-14
CmpDate: 2023-03-14

Freund EC, Haag SM, Haley B, et al (2023)

Optimized Nonviral Gene Disruption in Primary Murine and Human Myeloid Cells.

Methods in molecular biology (Clifton, N.J.), 2618:201-217.

Genetically engineered myeloid cells such as monocytes, macrophages, and dendritic cells have broad applications in basic and translational research. Their central roles in innate and adaptive immunity make them attractive as putative therapeutic cell products. However, efficient gene editing of primary myeloid cells presents unique challenges owing to their sensitivity to foreign nucleic acids and poor editing efficiencies using current methodologies (Hornung et al., Science 314:994-997, 2006; Coch et al., PLoS One 8:e71057, 2013; Bartok and Hartmann, Immunity 53:54-77, 2020; Hartmann, Adv Immunol 133:121-169, 2017; Bobadilla et al., Gene Ther 20:514-520, 2013; Schlee and Hartmann, Nat Rev Immunol 16:566-580, 2016; Leyva et al., BMC Biotechnol 11:13, 2011). This chapter describes nonviral CRISPR-mediated gene knockout in primary human and murine monocytes as well as monocyte-derived or bone marrow-derived macrophages and dendritic cells. Electroporation-mediated delivery of recombinant Cas9 complexed with synthetic guide RNAs can be applied for population-level disruption of single or multiple gene targets.

RevDate: 2023-03-14
CmpDate: 2023-03-14

Ueda J, Yamazaki T, H Funakoshi (2023)

Toward the Development of Epigenome Editing-Based Therapeutics: Potentials and Challenges.

International journal of molecular sciences, 24(5):.

The advancement in epigenetics research over the past several decades has led to the potential application of epigenome-editing technologies for the treatment of various diseases. In particular, epigenome editing is potentially useful in the treatment of genetic and other related diseases, including rare imprinted diseases, as it can regulate the expression of the epigenome of the target region, and thereby the causative gene, with minimal or no modification of the genomic DNA. Various efforts are underway to successfully apply epigenome editing in vivo, such as improving target specificity, enzymatic activity, and drug delivery for the development of reliable therapeutics. In this review, we introduce the latest findings, summarize the current limitations and future challenges in the practical application of epigenome editing for disease therapy, and introduce important factors to consider, such as chromatin plasticity, for a more effective epigenome editing-based therapy.

RevDate: 2023-03-14
CmpDate: 2023-03-14

Zhou J, Li Z, Li Y, et al (2023)

Effects of Different Gene Editing Modes of CRISPR/Cas9 on Soybean Fatty Acid Anabolic Metabolism Based on GmFAD2 Family.

International journal of molecular sciences, 24(5):.

Δ[12]-fatty acid dehydrogenase (FAD2) is the essential enzyme responsible for catalyzing the formation of linoleic acid from oleic acid. CRISPR/Cas9 gene editing technology has been an essential tool for molecular breeding in soybeans. To evaluate the most suitable type of gene editing in soybean fatty acid synthesis metabolism, this study selected five crucial enzyme genes of the soybean FAD2 gene family-GmFAD2-1A, GmFAD2-1B, GmFAD2-2A, GmFAD2-2B, and GmFAD2-2C-and created a CRISPR/Cas9-mediated single gene editing vector system. The results of Sanger sequencing showed that 72 transformed plants positive for T1 generation were obtained using Agrobacterium-mediated transformation, of which 43 were correctly edited plants, with the highest editing efficiency of 88% for GmFAD2-2A. The phenotypic analysis revealed that the oleic acid content of the progeny of GmFAD2-1A gene-edited plants had a higher increase of 91.49% when compared to the control JN18, and the rest of the gene-edited plants in order were GmFAD2-2A, GmFAD2-1B, GmFAD2-2C, and GmFAD2-2B. The analysis of gene editing type has indicated that base deletions greater than 2bp were the predominant editing type in all editing events. This study provides ideas for the optimization of CRISPR/Cas9 gene editing technology and the development of new tools for precise base editing in the future.

RevDate: 2023-03-14
CmpDate: 2023-03-14

Ivanov AV, Safenkova IV, Biketov SF, et al (2023)

Engineering of DNA Structures Attached to Magnetic Particles for Effective Trans- and Cis-Cleavage in Cas12-Based Biosensors.

International journal of molecular sciences, 24(5):.

Sequence-specific endonuclease Cas12-based biosensors have rapidly evolved as a strong tool to detect nucleic acids. Magnetic particles (MPs) with attached DNA structures could be used as a universal platform to manipulate the DNA-cleavage activity of Cas12. Here, we propose nanostructures of trans- and cis-DNA targets immobilized on the MPs. The main advantage of the nanostructures is a rigid double-stranded DNA adaptor that distances the cleavage site from the MP surface to ensure maximum Cas12 activity. Adaptors with different lengths were compared by detecting the cleavage by fluorescence and gel electrophoresis of the released DNA fragments. The length-dependent effects for cleavage on the MPs' surface were found both for cis- and trans-targets. For trans-DNA targets with a cleavable 15-dT tail, the results showed that the optimal range of the adaptor length was 120-300 bp. For cis-targets, we varied the length and location of the adaptor (at the PAM or spacer ends) to estimate the effect of the MP's surface on the PAM-recognition process or R-loop formation. The sequential arrangement of an adaptor, PAM, and a spacer was preferred and required the minimum adaptor length of 3 bp. Thus, with cis-cleavage, the cleavage site can be located closer to the surface of the MPs than with trans-cleavage. The findings provide solutions for efficient Cas12-based biosensors using surface-attached DNA structures.

RevDate: 2023-03-14
CmpDate: 2023-03-14

Wang W, Huang P, Dai W, et al (2023)

Application of Nicotinamide to Culture Medium Improves the Efficiency of Genome Editing in Hexaploid Wheat.

International journal of molecular sciences, 24(5):.

Histone acetylation is the earliest and most well-characterized of post-translation modifications. It is mediated by histone acetyltransferases (HAT) and histone deacetylases (HDAC). Histone acetylation could change the chromatin structure and status and further regulate gene transcription. In this study, nicotinamide, a histone deacetylase inhibitor (HDACi), was used to enhance the efficiency of gene editing in wheat. Transgenic immature and mature wheat embryos harboring a non-mutated GUS gene, the Cas9 and a GUS-targeting sgRNA were treated with nicotinamide in two concentrations (2.5 and 5 mM) for 2, 7, and 14 days in comparison with a no-treatment control. The nicotinamide treatment resulted in GUS mutations in up to 36% of regenerated plants, whereas no mutants were obtained from the non-treated embryos. The highest efficiency was achieved when treated with 2.5 mM nicotinamide for 14 days. To further validate the impact of nicotinamide treatment on the effectiveness of genome editing, the endogenous TaWaxy gene, which is responsible for amylose synthesis, was tested. Utilizing the aforementioned nicotinamide concentration to treat embryos containing the molecular components for editing the TaWaxy gene, the editing efficiency could be increased to 30.3% and 13.3%, respectively, for immature and mature embryos in comparison to the 0% efficiency observed in the control group. In addition, nicotinamide treatment during transformation progress could also improve the efficiency of genome editing approximately threefold in a base editing experiment. Nicotinamide, as a novel approach, may be employed to improve the editing efficacy of low-efficiency genome editing tools such as base editing and prime editing (PE) systems in wheat.

RevDate: 2023-03-14
CmpDate: 2023-03-14

Dou B, Zhang Y, Gao H, et al (2023)

CRISPR/Cas12a-based MUSCA-PEC strategy for HSV-1 assay.

Analytica chimica acta, 1250:340955.

In the photoelectrochemical sensing, constant potential excitation to get the photoelectrochemical signal is the main excitation signal mode. Novel method for photoelectrochemical signal obtaining is needed. Inspired by this ideal, a photoelectrochemical strategy for Herpes simplex virus (HSV-1) detection with multiple potential step chronoamperometry (MUSCA) pattern was fabricated using CRISPR/Cas12a cleavage coupled with entropy-driven target recycling. In the presence of target, HSV-1, the Cas12a was activated by the H1-H2 complex obtained by entropy-driven, then digesting the circular fragment of csRNA to expose single-stranded crRNA2 and alkaline phosphatase (ALP). The inactive Cas12a was self-assembled with crRNA2 and activated again with the help of assistant dsDNA. After multiple rounds of CRISPR/Cas12a cleavage and magnetic separation, MUSCA, as a signal amplifier, collected the enhanced photocurrent responses generated by catalyzed p-Aminophenol (p-AP). Different from the reported signal enhancement strategies based on photoactive nanomaterials and sensing mechanisms, MUSCA technique endowed the strategy with unique advantages of direct, fast and ultrasensitive. A superior detection limit of 3 aM toward HSV-1 was achieved. This strategy was successfully applied for HSV-1 detection in Human serum samples. The combination of MUSCA technique and CRISPR/Cas12a assay brings broader potential prospect for the detection of nucleic acids.

RevDate: 2023-03-14
CmpDate: 2023-03-14

Li M, Luo N, Liao X, et al (2023)

Proximity hybridization-regulated CRISPR/Cas12a-based dual signal amplification strategy for sensitive detection of circulating tumor DNA.

Talanta, 257:124395.

Circulating tumor DNA (ctDNA) is regarded as an ideal candidate biomarker for the non-invasive diagnosis of cancer. However, the lack of convenient and reliable detection methods for ctDNA restricts its clinical application. Herein, we developed a dual signal amplification strategy for sensitive detection of ctDNA based on hybridization chain reaction (HCR) and proximity hybridization-regulated CRISPR/Cas12a. The ctDNA initiates HCR through the continuous hybridization of two hairpin probes (H1 and H2), yielding long nicked double-stranded DNA nanowires composed of numerous split segments, which are successively connected to activate the trans-cleavage activity of CRISPR/Cas12a. In this case, the doubly labeled single-stranded DNA reporter can be cleaved to produce a strong fluorescent signal. Owing to the dual amplification of HCR and CRISPR/Cas12a, this strategy exhibits high sensitivity toward ctDNA with a low detection limit of 5.43 fM. Moreover, the proposed method was successfully applied for ctDNA detection in serum samples with satisfactory results, which has great potential in the clinical diagnosis of cancer.

RevDate: 2023-03-14
CmpDate: 2023-03-14

Zhang L, Bai T, H Wu (2023)

sgRNA-2wPSM: Identify sgRNAs on-target activity by combining two-window-based position specific mismatch and synthetic minority oversampling technique.

Computers in biology and medicine, 155:106489.

sgRNAs on-target activity prediction is a critical step in the CRISPR-Cas9 system. Due to its importance to RNA function research and genome editing application, some computational methods were introduced, treating it as a binary classification task or a regression task. Among these methods, sgRNA-PSM is a state-of-the-art method. In this work, we improved this method by proposing a new feature extraction method called two-window-based PSM, which divides the DNA sequences into two non-overlapping segments so as to extract different patterns in the two different segments. The two-window-based PSM were fed into Support Vector Machines (SVMs), and a new method called sgRNA-2wPSM was proposed. Furthermore, a new oversampling method called SCORE-SVM-SMOTE was proposed to solve the imbalanced training set problem based on the SVM-SMOTE algorithm. Results on the benchmark datasets indicated that sgRNA-2wPSM is superior to other methods.

RevDate: 2023-03-14
CmpDate: 2023-03-14

Liu D, Sun XM, Zhu L, et al (2023)

Using time-shared scanning optical tweezers assisted two-photon fluorescence imaging to establish a versatile CRISPR/Cas12a-mediated biosensor.

Biosensors & bioelectronics, 227:115158.

Based on the admirable precision to identify target nucleic acids and the particular trans-cleavage feature, CRISPR/Cas12a system is a useful means to further improve the sensing accuracy and the design flexibility of fluorescence biosensors. However, the current construction concepts still suffer from insufficient sensitivity, unsuitable for complicated real samples and limited detection species. In this work, much efforts are achieved to address these obstacles. At first, we adopt a microsphere sustained signal enrichment, under which a home-made time-shared scanning optical tweezers assisted fluorescence imaging is employed to guarantee a stable excitation and also realize multiflux measurement. Furthermore, by taking advantage of the low background merit of the near-infrared light excited two-photon fluorescence, a commendable anti-interference capability is endowed to operate in complex media. After utilizing a functional DNA (e.g. aptamer and DNAzyme) regulated mediation pathway to respond non-nucleic acid analytes (alpha fetal protein and Pb[2+]), the newly-established CRISPR/Cas12a-mediated fluorescence biosensor is found to display favorable assay performance. More importantly, our analytical methodology can act as a versatile and reliable toolbox in various applications such as disease diagnosis and environmental analysis, propelling the development of CRISPR system in biosensing field.

RevDate: 2023-03-14
CmpDate: 2023-03-14

Liu W, Zhu X, Li L, et al (2023)

Reining in Cas13a activity with N-terminal removable tags expands Cas13a based molecular sensing and enables precise gene interference.

Biosensors & bioelectronics, 227:115138.

Activation of Cas13 is exclusively dependent on crRNA-target RNA hybridization according to the canonical mode of Cas13 action. Upon activation Cas13 can cleave both target RNA and any surrounding RNA. The latter has been well adopted by therapeutic gene interference and biosensor development. This work for the first time, rationale designs and validates a multi-component controlled activation system of Cas13 by N-terminus tagging. A composite SUMO tag comprised of His, Twinstrep, and Smt3 tags fully suppresses target dependent activation of Cas13a by interfering with crRNA docking. The suppression releases upon proteases mediated proteolytic cleavage. The modular composition of the composite tag can be altered to fulfill customized response to alternative proteases. The biosensor SUMO-Cas13a is able to resolve a broad concentration range of protease Ulp1 with a calculated LOD of 48.8pg/μL in aqueous buffer. Further, in accordance with this finding Cas13a was successfully programmed to exert target gene knock down preferentially in SUMO protease high cell types. In summary the discovered regulatory component not only fulfills Cas13a based protease detection for the first time, but also delivers a novel strategy for multi-component controlled activation of Cas13a toward temporal and spacial precision.

RevDate: 2023-03-14
CmpDate: 2023-03-14

Wang X, Chen Y, Ma L, et al (2023)

An amplification-free CRISPR/Cas12a-based fluorescence assay for ultrasensitive detection of nuclease activity.

Talanta, 257:124329.

Nuclease, such as RNase H and DNase I, plays key roles in plenty of cellular processes and could be potential therapeutic target for drug development. It is necessary to establish rapid and simple-to-use methods to detect nuclease activity. Herein, we develop a Cas12a-based fluorescence assay without any nucleic acid amplification steps for ultrasensitive detection of RNase H or DNase I activity. By our design, the pre-assembled crRNA/ssDNA duplex triggered the cleavage of fluorescent probes in the presence of Cas12a enzymes. However, the crRNA/ssDNA duplex was selectively digested with the addition of RNase H or DNase I, which leaded to fluorescence intensity changes. Under optimized conditions, the method exhibited good analytical performance, achieving a limit of detection (LOD) as low as 0.0082 U/mL for RNase H and 0.13 U/mL for DNase I, respectively. The method was feasible for analysis of RNase H in human serum and cell lysates, as well as for screening of enzyme inhibitors. Moreover, it can be adopted to image RNase H activity in living cells. Together, this study provides a facile platform for nuclease detection and could be expanded for other biomedical research and clinical diagnostics.

RevDate: 2023-03-14
CmpDate: 2023-03-14

Zhao J, Tan Y, Jiang Y, et al (2023)

CRISPR/Cas9-mediated methoprene-tolerant 1 knockout results in precocious metamorphosis of beet armyworm (Spodoptera exigua) only at the late larval stage.

Insect molecular biology, 32(2):132-142.

Juvenile hormone (JH) controls almost every aspect of an insect, especially metamorphosis. Since RNA interference works on transcripts and is often insufficient in Lepidoptera, how JH affects larval development in these insects is not well studied. Using the CRISPR/Cas9 technique, we knocked out Spodoptera exigua methoprene-tolerant 1 (SeMet1) gene of beet armyworm by modifying two sites in the coding region. However, SeMet1 knockout did not affect egg hatch rate or larval development at L1-L3 stages. In contrast to the consistent five larval instars of the control group, L4 SeMet1 mutants began to show signs of precocious metamorphosis, that is, small patches of pupal cuticle. Most L4 and all L5 SeMet1 mutants died for failing to shed their mosaic cuticles. RNA-seq indicated that most genes encoding pupal cuticle proteins and chitinase genes were altered in SeMet1 mutant L4 larvae. SeKr-h1, a key transcription factor in JH action was significantly down-regulated in L3-L5 larvae, while SeBR-C, a pupal indicator was only upregulated in L4-L5 larvae. These results suggested that S. exigua larvae may initially develop independently of JH, and involve SeMet1 in transducing JH signalling, leading to controlled larval metamorphosis at the late larval stage. We believe our findings will enhance better understanding of JH regulation of larval development.

RevDate: 2023-03-13

Kogawa M, Nishikawa Y, Saeki T, et al (2023)

Revealing within-species diversity in uncultured human gut bacteria with single-cell long-read sequencing.

Frontiers in microbiology, 14:1133917.

Obtaining complete and accurate bacterial genomes is vital for studying the characteristics of uncultured bacteria. Single-cell genomics is a promising approach for the culture-independent recovery of bacterial genomes from individual cells. However, single-amplified genomes (SAGs) often have fragmented and incomplete sequences due to chimeric and biased sequences introduced during the genome amplification process. To address this, we developed a single-cell amplified genome long-read assembly (scALA) workflow to construct complete circular SAGs (cSAGs) from long-read single-cell sequencing data of uncultured bacteria. We used the SAG-gel platform, which is both cost-effective and high-throughput, to obtain hundreds of short-read and long-read sequencing data for specific bacterial strains. The scALA workflow generated cSAGs by repeated in silico processing for sequence bias reduction and contig assembly. From 12 human fecal samples, including two cohabitant groups, scALA generated 16 cSAGs of three specifically targeted bacterial species: Anaerostipes hadrus, Agathobacter rectalis, and Ruminococcus gnavus. We discovered strain-specific structural variations shared among cohabiting hosts, while all cSAGs of the same species showed high homology in aligned genomic regions. A. hadrus cSAGs exhibited 10 kbp-long phage insertions, various saccharide metabolic capabilities, and different CRISPR-Cas systems in each strain. The sequence similarity of A. hadrus genomes did not necessarily correspond with orthologous functional genes, while host geographical regionality seemed to be highly related to gene possession. scALA allowed us to obtain closed circular genomes of specifically targeted bacteria from human microbiota samples, leading to an understanding of within-species diversities, including structural variations and linking mobile genetic elements, such as phages, to hosts. These analyses provide insight into microbial evolution, the adaptation of the community to environmental changes, and interactions with hosts. cSAGs constructed using this method can expand bacterial genome databases and our understanding of within-species diversities in uncultured bacteria.

RevDate: 2023-03-13

Majeed Y, Zhu X, Zhang N, et al (2023)

Harnessing the role of mitogen-activated protein kinases against abiotic stresses in plants.

Frontiers in plant science, 14:932923.

Crop plants are vulnerable to various biotic and abiotic stresses, whereas plants tend to retain their physiological mechanisms by evolving cellular regulation. To mitigate the adverse effects of abiotic stresses, many defense mechanisms are induced in plants. One of these mechanisms is the mitogen-activated protein kinase (MAPK) cascade, a signaling pathway used in the transduction of extracellular stimuli into intercellular responses. This stress signaling pathway is activated by a series of responses involving MAPKKKs→MAPKKs→MAPKs, consisting of interacting proteins, and their functions depend on the collaboration and activation of one another by phosphorylation. These proteins are key regulators of MAPK in various crop plants under abiotic stress conditions and also related to hormonal responses. It is revealed that in response to stress signaling, MAPKs are characterized as multigenic families and elaborate the specific stimuli transformation as well as the antioxidant regulation system. This pathway is directed by the framework of proteins and stopping domains confer the related associates with unique structure and functions. Early studies of plant MAPKs focused on their functions in model plants. Based on the results of whole-genome sequencing, many MAPKs have been identified in plants, such as Arbodiposis, tomato, potato, alfalfa, poplar, rice, wheat, maize, and apple. In this review, we summarized the recent work on MAPK response to abiotic stress and the classification of MAPK cascade in crop plants. Moreover, we highlighted the modern research methodologies such as transcriptomics, proteomics, CRISPR/Cas technology, and epigenetic studies, which proposed, identified, and characterized the novel genes associated with MAPKs and their role in plants under abiotic stress conditions. In-silico-based identification of novel MAPK genes also facilitates future research on MAPK cascade identification and function in crop plants under various stress conditions.

RevDate: 2023-03-13

Tiwari JK, Singh AK, TK Behera (2023)

CRISPR/Cas genome editing in tomato improvement: Advances and applications.

Frontiers in plant science, 14:1121209.

The narrow genetic base of tomato poses serious challenges in breeding. Hence, with the advent of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein9 (CRISPR/Cas9) genome editing, fast and efficient breeding has become possible in tomato breeding. Many traits have been edited and functionally characterized using CRISPR/Cas9 in tomato such as plant architecture and flower characters (e.g. leaf, stem, flower, male sterility, fruit, parthenocarpy), fruit ripening, quality and nutrition (e.g., lycopene, carotenoid, GABA, TSS, anthocyanin, shelf-life), disease resistance (e.g. TYLCV, powdery mildew, late blight), abiotic stress tolerance (e.g. heat, drought, salinity), C-N metabolism, and herbicide resistance. CRISPR/Cas9 has been proven in introgression of de novo domestication of elite traits from wild relatives to the cultivated tomato and vice versa. Innovations in CRISPR/Cas allow the use of online tools for single guide RNA design and multiplexing, cloning (e.g. Golden Gate cloning, GoldenBraid, and BioBrick technology), robust CRISPR/Cas constructs, efficient transformation protocols such as Agrobacterium, and DNA-free protoplast method for Cas9-gRNAs ribonucleoproteins (RNPs) complex, Cas9 variants like PAM-free Cas12a, and Cas9-NG/XNG-Cas9, homologous recombination (HR)-based gene knock-in (HKI) by geminivirus replicon, and base/prime editing (Target-AID technology). This mini-review highlights the current research advances in CRISPR/Cas for fast and efficient breeding of tomato.

RevDate: 2023-03-12

Duan W, Urani E, MP Mattson (2023)

The potential of gene editing for Huntington's disease.

Trends in neurosciences pii:S0166-2236(23)00046-2 [Epub ahead of print].

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by a trinucleotide repeat expansion in the huntingtin gene resulting in long stretches of polyglutamine repeats in the huntingtin protein. The disease involves progressive degeneration of neurons in the striatum and cerebral cortex resulting in loss of control of motor function, psychiatric problems, and cognitive deficits. There are as yet no treatments that can slow disease progression in HD. Recent advances in gene editing using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) systems and demonstrations of their ability to correct gene mutations in animal models of a range of diseases suggest that gene editing may prove effective in preventing or ameliorating HD. Here we describe (i) potential CRISPR-Cas designs and cellular delivery methods for the correction of mutant genes that cause inherited diseases, and (ii) recent preclinical findings demonstrating the efficacy of such gene-editing approaches in animal models, with a focus on HD.

RevDate: 2023-03-11

Matsumoto D, Kishi K, Matsugi E, et al (2023)

Cas9-Geminin and Cdt1-fused anti-CRISPR protein synergistically increase editing accuracy.

FEBS letters [Epub ahead of print].

Genome editing with CRISPR-Cas9, particularly for therapeutic purposes, should be accomplished via the homology-directed repair (HDR) pathway, which exhibits greater precision than other pathways. However, one of the issues to be solved is that genome editing efficiency with HDR is generally low. A Streptococcus pyogenes Cas9 (SpyCas9) fusion with human Geminin (Cas9-Gem) reportedly increases HDR efficiency slightly. In contrast, we found that regulation of SpyCas9 activity with an anti-CRISPR protein (AcrIIA4) fused to Chromatin licensing and DNA replication factor 1 (Cdt1) significantly increases HDR efficiency and reduces off-target effects. Here, another anti-CRISPR protein, AcrIIA5, was applied, and the combined use of Cas9-Gem and Anti-CRISPR+Cdt1 showed synergistic enhancement of HDR efficiency. The method may be applicable to various anti-CRISPR/CRISPR-Cas combinations.

RevDate: 2023-03-13

Kong Q, Li J, Wang S, et al (2023)

Combination of Hairy Root and Whole-Plant Transformation Protocols to Achieve Efficient CRISPR/Cas9 Genome Editing in Soybean.

Plants (Basel, Switzerland), 12(5):.

The new gene-editing technology CRISPR/Cas system has been widely used for genome engineering in various organisms. Since the CRISPR/Cas gene-editing system has a certain possibility of low efficiency and the whole plant transformation of soybean is time-consuming and laborious, it is important to evaluate the editing efficiency of designed CRISPR constructs before the stable whole plant transformation process starts. Here, we provide a modified protocol for generating transgenic hairy soybean roots to assess the efficiency of guide RNA (gRNA) sequences of the CRISPR/Cas constructs within 14 days. The cost- and space-effective protocol was first tested in transgenic soybean harboring the GUS reporter gene for the efficiency of different gRNA sequences. Targeted DNA mutations were detected in 71.43-97.62% of the transgenic hairy roots analyzed as evident by GUS staining and DNA sequencing of the target region. Among the four designed gene-editing sites, the highest editing efficiency occurred at the 3' terminal of the GUS gene. In addition to the reporter gene, the protocol was tested for the gene-editing of 26 soybean genes. Among the gRNAs selected for stable transformation, the editing efficiency of hairy root transformation and stable transformation ranged from 5% to 88.8% and 2.7% to 80%, respectively. The editing efficiencies of stable transformation were positively correlated with those of hairy root transformation with a Pearson correlation coefficient (r) of 0.83. Our results demonstrated that soybean hairy root transformation could rapidly assess the efficiency of designed gRNA sequences on genome editing. This method can not only be directly applied to the functional study of root-specific genes, but more importantly, it can be applied to the pre-screening of gRNA in CRISPR/Cas gene editing.

RevDate: 2023-03-13

Liu B, Song W, Wang L, et al (2023)

dCas9-BE3 and dCas12a-BE3 Systems Mediated Base Editing in Kiwifruit Canker Causal Agent Pseudomonas syringae pv. actinidiae.

International journal of molecular sciences, 24(5):.

Pseudomonas syringae pv. actinidiae (Psa) causes bacterial canker of kiwifruit with heavy economic losses. However, little is known about the pathogenic genes of Psa. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas-mediated genome editing technology has dramatically facilitated the characterization of gene function in various organisms. However, CRISPR genome editing could not be efficiently employed in Psa due to lacking homologous recombination repair. The base editor (BE) system, which depends on CRISPR/Cas, directly induces single nucleoside C to T without homology recombination repair. Here, we used dCas9-BE3 and dCas12a-BE3 systems to create substitutions of C to T and to convert CAG/CAA/CGA codons to stop codons (TAG/TAA/TGA) in Psa. The dCas9-BE3 system-induced single C-to-T conversion frequency of 3 to 10 base positions ranged from 0% to 100%, with a mean of 77%. The dCas12a-BE3 system-induced single C-to-T conversion frequency of 8 to 14 base positions in the spacer region ranged from 0% to 100%, with a mean of 76%. In addition, a relatively saturated Psa gene knockout system covering more than 95% of genes was developed based on dCas9-BE3 and dCas12a-BE3, which could knock out two or three genes at the same time in the Psa genome. We also found that hopF2 and hopAO2 were involved in the Psa virulence of kiwifruit. The HopF2 effector can potentially interact with proteins such as RIN, MKK5, and BAK1, while the HopAO2 effector can potentially interact with the EFR protein to reduce the host's immune response. In conclusion, for the first time, we established a PSA.AH.01 gene knockout library that may promote research on elucidating the gene function and pathogenesis of Psa.

RevDate: 2023-03-13

Wang Y, Fan H, Y Tong (2023)

Unveil the Secret of the Bacteria and Phage Arms Race.

International journal of molecular sciences, 24(5):.

Bacteria have developed different mechanisms to defend against phages, such as preventing phages from being adsorbed on the surface of host bacteria; through the superinfection exclusion (Sie) block of phage's nucleic acid injection; by restricting modification (R-M) systems, CRISPR-Cas, aborting infection (Abi) and other defense systems to interfere with the replication of phage genes in the host; through the quorum sensing (QS) enhancement of phage's resistant effect. At the same time, phages have also evolved a variety of counter-defense strategies, such as degrading extracellular polymeric substances (EPS) that mask receptors or recognize new receptors, thereby regaining the ability to adsorb host cells; modifying its own genes to prevent the R-M systems from recognizing phage genes or evolving proteins that can inhibit the R-M complex; through the gene mutation itself, building nucleus-like compartments or evolving anti-CRISPR (Acr) proteins to resist CRISPR-Cas systems; and by producing antirepressors or blocking the combination of autoinducers (AIs) and its receptors to suppress the QS. The arms race between bacteria and phages is conducive to the coevolution between bacteria and phages. This review details bacterial anti-phage strategies and anti-defense strategies of phages and will provide basic theoretical support for phage therapy while deeply understanding the interaction mechanism between bacteria and phages.

RevDate: 2023-03-13
CmpDate: 2023-03-13

Pavlou S, Foskolou S, Patikas N, et al (2023)

CRISPR-Cas9 genetic screen leads to the discovery of L-Moses, a KAT2B inhibitor that attenuates Tunicamycin-mediated neuronal cell death.

Scientific reports, 13(1):3934.

Accumulation of aggregated and misfolded proteins, leading to endoplasmic reticulum stress and activation of the unfolded protein response, is a hallmark of several neurodegenerative disorders, including Alzheimer's and Parkinson's disease. Genetic screens are powerful tools that are proving invaluable in identifying novel modulators of disease associated processes. Here, we performed a loss-of-function genetic screen using a human druggable genome library, followed by an arrayed-screen validation, in human iPSC-derived cortical neurons. We identified and genetically validated 13 genes, whose knockout was neuroprotective against Tunicamycin, a glycoprotein synthesis inhibitor widely used to induce endoplasmic reticulum stress. We also demonstrated that pharmacological inhibition of KAT2B, a lysine acetyltransferase identified by our genetic screens, by L-Moses, attenuates Tunicamycin-mediated neuronal cell death and activation of CHOP, a key pro-apoptotic member of the unfolded protein response in both cortical and dopaminergic neurons. Follow-up transcriptional analysis suggested that L-Moses provided neuroprotection by partly reversing the transcriptional changes caused by Tunicamycin. Finally, L-Moses treatment attenuated total protein levels affected by Tunicamycin, without affecting their acetylation profile. In summary, using an unbiased approach, we identified KAT2B and its inhibitor, L-Moses, as potential therapeutic targets for neurodegenerative diseases.

RevDate: 2023-03-13
CmpDate: 2023-03-13

Komori T, Hata S, Mabuchi A, et al (2023)

A CRISPR-del-based pipeline for complete gene knockout in human diploid cells.

Journal of cell science, 136(6):.

The advance of CRISPR/Cas9 technology has enabled us easily to generate gene knockout cell lines by introducing insertion-deletion mutations (indels) at the target site via the error-prone non-homologous end joining repair system. Frameshift-promoting indels can disrupt gene functions by generation of a premature stop codon. However, there is growing evidence that targeted genes are not always knocked out by the indel-based gene disruption. Here, we established a pipeline of CRISPR-del, which induces a large chromosomal deletion by cutting two different target sites, to perform 'complete' gene knockout efficiently in human diploid cells. Quantitative analyses show that the frequency of gene deletion with this approach is much higher than that of conventional CRISPR-del methods. The lengths of the deleted genomic regions demonstrated in this study are longer than those of 95% of the human protein-coding genes. Furthermore, the pipeline enabled the generation of a model cell line having a bi-allelic cancer-associated chromosomal deletion. Overall, these data lead us to propose that the CRISPR-del pipeline is an efficient and practical approach for producing 'complete' gene knockout cell lines in human diploid cells.

RevDate: 2023-03-13
CmpDate: 2023-03-13

Dickson A, Mullooly N, Serrano A, et al (2023)

Highly scalable arrayed CRISPR mediated gene silencing in primary lung small airway epithelial cells.

SLAS discovery : advancing life sciences R & D, 28(2):29-35.

Small airway epithelial cells (SAECs) play a central role in the pathogenesis of lung diseases and are now becoming a crucial cellular model for target identification and validation in drug discovery. However, primary cell lines such as SAECs are often difficult to transfect using traditional lipofection methods; therefore, gene editing using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is often carried out through ribonucleoprotein (RNP) electroporation. Here we have established a robust, scalable, and automated arrayed CRISPR nuclease (CRISPRn) screening workflow for SAECs which can be combined with a myriad of disease-specific endpoint assays.

RevDate: 2023-03-13
CmpDate: 2023-03-13

Hardouin G, Antoniou P, Martinucci P, et al (2023)

Adenine base editor-mediated correction of the common and severe IVS1-110 (G>A) β-thalassemia mutation.

Blood, 141(10):1169-1179.

β-Thalassemia (BT) is one of the most common genetic diseases worldwide and is caused by mutations affecting β-globin production. The only curative treatment is allogenic hematopoietic stem/progenitor cells (HSPCs) transplantation, an approach limited by compatible donor availability and immunological complications. Therefore, transplantation of autologous, genetically-modified HSPCs is an attractive therapeutic option. However, current gene therapy strategies based on the use of lentiviral vectors are not equally effective in all patients and CRISPR/Cas9 nuclease-based strategies raise safety concerns. Thus, base editing strategies aiming to correct the genetic defect in patients' HSPCs could provide safe and effective treatment. Here, we developed a strategy to correct one of the most prevalent BT mutations (IVS1-110 [G>A]) using the SpRY-ABE8e base editor. RNA delivery of the base editing system was safe and led to ∼80% of gene correction in the HSPCs of patients with BT without causing dangerous double-strand DNA breaks. In HSPC-derived erythroid populations, this strategy was able to restore β-globin production and correct inefficient erythropoiesis typically observed in BT both in vitro and in vivo. In conclusion, this proof-of-concept study paves the way for the development of a safe and effective autologous gene therapy approach for BT.

RevDate: 2023-03-13
CmpDate: 2023-03-13

Jin S, Lin Q, Gao Q, et al (2023)

Optimized prime editing in monocot plants using PlantPegDesigner and engineered plant prime editors (ePPEs).

Nature protocols, 18(3):831-853.

Prime editors (PEs), which can install desired base edits without donor DNA or double-strand breaks, have been used in plants and can, in principle, accelerate crop improvement and breeding. However, their editing efficiency in plants is generally low. Optimizing the prime editing guide RNA (pegRNA) by designing the sequence on the basis of melting temperature, using dual-pegRNAs and engineering PEs have all been shown to enhance PE efficiency. In addition, an automated pegRNA design platform, PlantPegDesigner, has been developed on the basis of rice prime editing experimental data. In this protocol, we present detailed protocols for designing and optimizing pegRNAs using PlantPegDesigner, constructing engineered plant PE vectors with enhanced editing efficiency for prime editing, evaluating prime editing efficiencies using a reporter system and comparing the effectiveness and byproducts of PEs by deep amplicon sequencing. Using this protocol, researchers can construct optimized pegRNAs for prime editing in 4-7 d and obtain prime-edited rice or wheat plants within 3 months.

RevDate: 2023-03-13
CmpDate: 2023-03-13

Hu X, Zhang B, Li X, et al (2023)

The application and progression of CRISPR/Cas9 technology in ophthalmological diseases.

Eye (London, England), 37(4):607-617.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) system is an adaptive immune defence system that has gradually evolved in bacteria and archaea to combat invading viruses and exogenous DNA. Advances in technology have enabled researchers to enhance their understanding of the immune process in vivo and its potential for use in genome editing. Thus far, applications of CRISPR/Cas9 genome editing technology in ophthalmology have included gene therapy for corneal dystrophy, glaucoma, congenital cataract, Leber's congenital amaurosis, retinitis pigmentosa, Usher syndrome, fundus neovascular disease, proliferative vitreoretinopathy, retinoblastoma and other eye diseases. Additionally, the combination of CRISPR/Cas9 genome editing technology with adeno-associated virus vector and inducible pluripotent stem cells provides further therapeutic avenues for the treatment of eye diseases. Nonetheless, many challenges remain in the development of clinically feasible retinal genome editing therapy. This review discusses the development, as well as mechanism of CRISPR/Cas9 and its applications and challenges in gene therapy for eye diseases.

RevDate: 2023-03-10

Elmore JR, Dexter GN, Baldino H, et al (2023)

High-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration.

Science advances, 9(10):eade1285.

Efficient genome engineering is critical to understand and use microbial functions. Despite recent development of tools such as CRISPR-Cas gene editing, efficient integration of exogenous DNA with well-characterized functions remains limited to model bacteria. Here, we describe serine recombinase-assisted genome engineering, or SAGE, an easy-to-use, highly efficient, and extensible technology that enables selection marker-free, site-specific genome integration of up to 10 DNA constructs, often with efficiency on par with or superior to replicating plasmids. SAGE uses no replicating plasmids and thus lacks the host range limitations of other genome engineering technologies. We demonstrate the value of SAGE by characterizing genome integration efficiency in five bacteria that span multiple taxonomy groups and biotechnology applications and by identifying more than 95 heterologous promoters in each host with consistent transcription across environmental and genetic contexts. We anticipate that SAGE will rapidly expand the number of industrial and environmental bacteria compatible with high-throughput genetics and synthetic biology.

RevDate: 2023-03-10

Yang K, Qian J, Zhang C, et al (2023)

Biogenic materials for CRISPR delivery and therapeutics.

Biomaterials science [Epub ahead of print].

CRISPR, as an emerging gene-editing technology, has been widely used in multidisciplinary fields, including genetic diseases and some cancers. However, it remains a challenge to efficiently deliver CRISPR for safe and efficient genome editing. Currently, biomimetic materials have become an attractive delivery strategy for CRISPR-mediated genome editing due to their low immunogenicity and application safety. The biomimetic materials delivery is involved in the improvement of cellular uptake of nanoparticle vectors, and the gene editing efficiency. In this review, we summarize the current delivery strategies of CRISPR/Cas systems based on biogenic materials such as viruses, bacteria, cells, bioactive substances, etc., focusing on the potential applications in disease research and therapy. Finally, the prospects and limitations of CRISPR-based systems in therapeutics are discussed.

RevDate: 2023-03-11

Petersen B (2023)

Editorial: Genetic engineering in farm animals.

Frontiers in genetics, 14:1155201.

RevDate: 2023-03-11

Hu M, Bao W, Peng Q, et al (2023)

Metabolic engineering of Zymomonas mobilis for co-production of D-lactic acid and ethanol using waste feedstocks of molasses and corncob residue hydrolysate.

Frontiers in bioengineering and biotechnology, 11:1135484.

Lactate is the precursor for polylactide. In this study, a lactate producer of Z. mobilis was constructed by replacing ZMO0038 with LmldhA gene driven by a strong promoter PadhB, replacing ZMO1650 with native pdc gene driven by Ptet, and replacing native pdc with another copy of LmldhA driven by PadhB to divert carbon from ethanol to D-lactate. The resultant strain ZML-pdc-ldh produced 13.8 ± 0.2 g/L lactate and 16.9 ± 0.3 g/L ethanol using 48 g/L glucose. Lactate production of ZML-pdc-ldh was further investigated after fermentation optimization in pH-controlled fermenters. ZML-pdc-ldh produced 24.2 ± 0.6 g/L lactate and 12.9 ± 0.8 g/L ethanol as well as 36.2 ± 1.0 g/L lactate and 40.3 ± 0.3 g/L ethanol, resulting in total carbon conversion rate of 98.3% ± 2.5% and 96.2% ± 0.1% with final product productivity of 1.9 ± 0.0 g/L/h and 2.2 ± 0.0 g/L/h in RMG5 and RMG12, respectively. Moreover, ZML-pdc-ldh produced 32.9 ± 0.1 g/L D-lactate and 27.7 ± 0.2 g/L ethanol as well as 42.8 ± 0.0 g/L D-lactate and 53.1 ± 0.7 g/L ethanol with 97.1% ± 0.0% and 99.1% ± 0.8% carbon conversion rate using 20% molasses or corncob residue hydrolysate, respectively. Our study thus demonstrated that it is effective for lactate production by fermentation condition optimization and metabolic engineering to strengthen heterologous ldh expression while reducing the native ethanol production pathway. The capability of recombinant lactate-producer of Z. mobilis for efficient waste feedstock conversion makes it a promising biorefinery platform for carbon-neutral biochemical production.

RevDate: 2023-03-11

Li ZH, Wang J, Xu JP, et al (2023)

Recent advances in CRISPR-based genome editing technology and its applications in cardiovascular research.

Military Medical Research, 10(1):12.

The rapid development of genome editing technology has brought major breakthroughs in the fields of life science and medicine. In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing toolbox has been greatly expanded, not only with emerging CRISPR-associated protein (Cas) nucleases, but also novel applications through combination with diverse effectors. Recently, transposon-associated programmable RNA-guided genome editing systems have been uncovered, adding myriads of potential new tools to the genome editing toolbox. CRISPR-based genome editing technology has also revolutionized cardiovascular research. Here we first summarize the advances involving newly identified Cas orthologs, engineered variants and novel genome editing systems, and then discuss the applications of the CRISPR-Cas systems in precise genome editing, such as base editing and prime editing. We also highlight recent progress in cardiovascular research using CRISPR-based genome editing technologies, including the generation of genetically modified in vitro and animal models of cardiovascular diseases (CVD) as well as the applications in treating different types of CVD. Finally, the current limitations and future prospects of genome editing technologies are discussed.

RevDate: 2023-03-09

Hwang S, KL Maxwell (2023)

Diverse mechanisms of CRISPR-Cas9 inhibition by type II anti-CRISPR proteins.

Journal of molecular biology pii:S0022-2836(23)00097-9 [Epub ahead of print].

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) systems provide bacteria and archaea with an adaptive immune response against invasion by mobile genetic elements like phages, plasmids, and transposons. These systems have been repurposed as very powerful biotechnological tools for gene editing applications in both bacterial and eukaryotic systems. The discovery of natural off-switches for CRISPR-Cas systems, known as anti-CRISPR proteins, provided a mechanism for controlling CRISPR-Cas activity and opened avenues for the development of more precise editing tools. In this review, we focus on the inhibitory mechanisms of anti-CRISPRs that are active against type II CRISPR-Cas systems and briefly discuss their biotechnological applications.

RevDate: 2023-03-09

Yang H, Ledesma-Amaro R, Gao H, et al (2023)

CRISPR-based biosensors for pathogenic biosafety.

Biosensors & bioelectronics, 228:115189 pii:S0956-5663(23)00131-8 [Epub ahead of print].

Pathogenic biosafety is a worldwide concern. Tools for analyzing pathogenic biosafety, that are precise, rapid and field-deployable, are highly demanded. Recently developed biotechnological tools, especially those utilizing CRISPR/Cas systems which can couple with nanotechnologies, have enormous potential to achieve point-of-care (POC) testing for pathogen infection. In this review, we first introduce the working principle of class II CRISPR/Cas system for detecting nucleic acid and non-nucleic acid biomarkers, and highlight the molecular assays that leverage CRISPR technologies for POC detection. We summarize the application of CRISPR tools in detecting pathogens, including pathogenic bacteria, viruses, fungi and parasites and their variants, and highlight the profiling of pathogens' genotypes or phenotypes, such as the viability, and drug-resistance. In addition, we discuss the challenges and opportunities of CRISPR-based biosensors in pathogenic biosafety analysis.

RevDate: 2023-03-07

Yau WW, Chen GB, Zhou J, et al (2023)

Nicotinamide riboside rescues dysregulated glycolysis and fatty acid β-oxidation in a human hepatic cell model of Citrin deficiency.

Human molecular genetics pii:7070577 [Epub ahead of print].

Citrin Deficiency (CD) is an inborn error of metabolism caused by loss-of-function of the mitochondrial aspartate/glutamate transporter, CITRIN, which is involved in both the urea cycle and malate aspartate shuttle. Patients with CD develop hepatosteatosis and hyperammonemia but there is no effective therapy for CD. Currently, there are no animal models that faithfully recapitulate the human CD phenotype. Accordingly, we generated a CITRIN knockout HepG2 cell line using CRISPR/Cas 9 genome editing technology to study metabolic and cell signaling defects in CD. CITRIN KO cells showed increased ammonia accumulation, higher cytosolic NADH/NAD+ ratio and reduced glycolysis. Surprisingly, these cells showed impaired fatty acid metabolism and mitochondrial activity. CITRIN KO cells also displayed increased cholesterol and bile acid metabolism resembling those observed in CD patients. Remarkably, normalizing cytosolic NADH:NAD+ ratio by nicotinamide riboside (NR) increased glycolysis and fatty acid oxidation but had no effect on the hyperammonemia suggesting the urea cycle defect was independent of the aspartate/malate shuttle defect of CD. The correction of glycolysis and fatty acid metabolism defects in CITRIN KO cells by reducing cytoplasmic NADH:NAD+ levels suggests this may be a novel strategy to treat some of the metabolic defects of CD and other mitochondrial diseases.

RevDate: 2023-03-09
CmpDate: 2023-03-09

Chauhan VP, Sharp PA, R Langer (2023)

Altered DNA repair pathway engagement by engineered CRISPR-Cas9 nucleases.

Proceedings of the National Academy of Sciences of the United States of America, 120(11):e2300605120.

CRISPR-Cas9 introduces targeted DNA breaks that engage competing DNA repair pathways, producing a spectrum of imprecise insertion/deletion mutations (indels) and precise templated mutations (precise edits). The relative frequencies of these pathways are thought to primarily depend on genomic sequence and cell state contexts, limiting control over mutational outcomes. Here, we report that engineered Cas9 nucleases that create different DNA break structures engage competing repair pathways at dramatically altered frequencies. We accordingly designed a Cas9 variant (vCas9) that produces breaks which suppress otherwise dominant nonhomologous end-joining (NHEJ) repair. Instead, breaks created by vCas9 are predominantly repaired by pathways utilizing homologous sequences, specifically microhomology-mediated end-joining (MMEJ) and homology-directed repair (HDR). Consequently, vCas9 enables efficient precise editing through HDR or MMEJ while suppressing indels caused by NHEJ in dividing and nondividing cells. These findings establish a paradigm of targeted nucleases custom-designed for specific mutational applications.

RevDate: 2023-03-07

Zha MJ, Cai CE, PM He (2023)

Outlook on the Security and Potential Improvements of CRISPR-Cas9.

Molecular biotechnology [Epub ahead of print].

Gene editing technology is regarded as a good news to save patients with genetic diseases because of its significant function of specifically changing genetic information. From zinc-finger proteins to transcription activator-like effector protein nucleases gene editing tools are constantly updated. At the same time, scientists are constantly developing a variety of new gene editing therapy strategies, in order to promote gene editing therapy from various aspects and realize the maturity of the technology as soon as possible. In 2016, CRISPR-Cas9-mediated CAR-T therapy was the first to enter the clinical trial stage, indicating that the use of CRISPR-Cas system as the blade of the genetic lancet to save patients is officially on the schedule. The first challenge to achieve this exciting goal is to improve the security of the technology. This review will introduce the gene security issues faced by the CRISPR system as a clinical treatment tool, the current safer delivery methods and the newly developed CRISPR editing tools with higher precision. Many reviews summarize the means of improving the security of gene editing therapy and the comprehensive delivery method, while few articles focus on the threat of gene editing to the genomic security of the treatment target. Therefore, this review focuses on the risks brought by gene editing therapy to the patient genome, which provides a broader perspective for exploring and improving the security of gene editing therapy from two aspects of delivery system and CRISPR editing tools.

RevDate: 2023-03-09
CmpDate: 2023-03-09

Popescu B, K Shannon (2023)

Sidestepping SHP2 inhibition.

The Journal of experimental medicine, 220(5):.

Allosteric SHP2 inhibitors are a novel class of compounds that target hyperactive Ras/Mitogen Activated Protein Kinase (MAPK) signaling. In this issue of JEM, Wei et al. (2023. J. Exp. Med. report a genome-wide CRISPR/Cas9 knockout screen that uncovered novel mechanisms of adaptive resistance to pharmacologic inhibition of SHP2.

RevDate: 2023-03-07

Hernandes-Lopes J, Yassitepe JECT, Koltun A, et al (2023)

Genome editing in maize: Toward improving complex traits in a global crop.

Genetics and molecular biology, 46(1 Suppl 1):e20220217.

Recent advances in genome editing have enormously enhanced the effort to develop biotechnology crops for more sustainable food production. CRISPR/Cas, the most versatile genome-editing tool, has shown the potential to create genome modifications that range from gene knockout and gene expression pattern modulations to allele-specific changes in order to design superior genotypes harboring multiple improved agronomic traits. However, a frequent bottleneck is the delivery of CRISPR/Cas to crops that are less amenable to transformation and regeneration. Several technologies have recently been proposed to overcome transformation recalcitrance, including HI-Edit/IMGE and ectopic/transient expression of genes encoding morphogenic regulators. These technologies allow the eroding of the barriers that make crops inaccessible for genome editing. In this review, we discuss the advances in genome editing in crops with a particular focus on the use of technologies to improve complex traits such as water use efficiency, drought stress, and yield in maize.

RevDate: 2023-03-07

Punetha M, Saini S, Chaudhary S, et al (2023)

Induced Pluripotent Stem Cells in the Era of Precise Genome Editing.

Current stem cell research & therapy pii:CSCR-EPUB-130037 [Epub ahead of print].

Genome editing has enhanced our ability to understand the role of genetics in a number of diseases by facilitating the development of more precise cellular and animal models to study pathophysiological processes. These advances have shown extraordinary promise in a multitude of areas, from basic research to applied bioengineering and biomedical research. Induced pluripotent stem cells (iPSCs) are known for their high replicative capacity and are excellent targets for genetic manipulation as they can be clonally expanded from a single cell without compromising their pluripotency. Clustered, regularly interspaced short palindromic repeats (CRISPR) and CRISPR/Cas RNA-guided nucleases have rapidly become the method of choice for gene editing due to their high specificity, simplicity, low cost, and versatility. Coupling the cellular versatility of iPSCs differentiation with CRISPR/Cas9-mediated genome editing technology can be an effective experimental technique for providing new insights into the therapeutic use of this technology. However, before using these techniques for gene therapy, their therapeutic safety and efficacy following models need to be assessed. In this review, we cover the remarkable progress that has been made in the use of genome editing tools in iPSCs, their applications in disease research and gene therapy as well as the hurdles that remain in the actual implementation of CRISPR/Cas systems.

RevDate: 2023-03-09
CmpDate: 2023-03-08

Park J, Cui G, Lee H, et al (2023)

CRISPR/Cas9 mediated specific ablation of vegfa in retinal pigment epithelium efficiently regresses choroidal neovascularization.

Scientific reports, 13(1):3715.

The CRISPR/Cas9 system easily edits target genes in various organisms and is used to treat human diseases. In most therapeutic CRISPR studies, ubiquitously expressed promoters, such as CMV, CAG, and EF1α, are used; however, gene editing is sometimes necessary only in specific cell types relevant to the disease. Therefore, we aimed to develop a retinal pigment epithelium (RPE)-specific CRISPR/Cas9 system. We developed a CRISPR/Cas9 system that operates only in retinal pigment epithelium (RPE) by expressing Cas9 under the RPE-specific vitelliform macular dystrophy 2 promoter (pVMD2). This RPE-specific CRISPR/pVMD2-Cas9 system was tested in human retinal organoid and mouse model. We confirmed that this system works specifically in the RPE of human retinal organoids and mouse retina. In addition, the RPE-specific Vegfa ablation using the novel CRISPR-pVMD2-Cas9 system caused regression of choroidal neovascularization (CNV) without unwanted knock-out in the neural retina in laser-induced CNV mice, which is a widely used animal model of neovascular age-related macular degeneration. RPE-specific Vegfa knock-out (KO) and ubiquitous Vegfa KO were comparable in the efficient regression of CNV. The promoter substituted, cell type-specific CRISPR/Cas9 systems can be used in specific 'target cell' therapy, which edits genes while reducing unwanted off- 'target cell' effects.

RevDate: 2023-03-09
CmpDate: 2023-03-09

Jiang Y, Zheng C, Jin M, et al (2023)

An Ultrasensitive Colorimetric Foodborne Pathogenic Detection Method Using a CRISPR/Cas12a Mediated Strand Displacement/Hybridization Chain Reaction.

Journal of agricultural and food chemistry, 71(9):4193-4200.

Accurate, rapid, and sensitive pathogenic detections play an important role in food safety. Herein, we developed a novel CRISPR/Cas12a mediated strand displacement/hybridization chain reaction (CSDHCR) nucleic acid assay for foodborne pathogenic colorimetric detection. A biotinylated DNA toehold is coupled on avidin magnetic beads and acts as an initiator strand to trigger the SDHCR. The SDHCR amplification allowed the formation of long hemin/G-quadruplex-based DNAzyme products to catalyze the TMB-H2O2 reaction. In the presence of the DNA targets, the trans-cleavage activity of CRISPR/Cas12a was activated to cleave the initiator DNA, resulting in the failure of SDHCR and no color change. Under optimal conditions, the CSDHCR has a satisfactory linear detection of DNA targets with a regression equation Y = 0.0531*X - 0.0091 (R[2] = 0.9903) in the range of 10 fM to 1 nM, and the limit of detection was determined as 4.54 fM. In addition, Vibrio vulnificus, one foodborne pathogen, was used to verify the practical application of the method, and it showed satisfactory specificity and sensitivity with a limit of detection at 1.0 × 10[0] CFU/mL coupling with recombinase polymerase amplification. Our proposed CSDHCR biosensor could be a promising alternative method for ultrasensitive and visual detection of nucleic acids and the practical application of foodborne pathogens.

RevDate: 2023-03-09
CmpDate: 2023-03-09

Xu Y, Tian N, Shi H, et al (2023)

A Split CRISPR/Cas13b System for Conditional RNA Regulation and Editing.

Journal of the American Chemical Society, 145(9):5561-5569.

The CRISPR/Cas13b system has been demonstrated as a robust tool for versatile RNA studies and relevant applications. New strategies enabling precise control of Cas13b/dCas13b activities and minimal interference with native RNA activities will further facilitate the understanding and regulation of RNA functions. Here, we engineered a split Cas13b system that can be conditionally activated and deactivated under the induction of abscisic acid (ABA), which achieved the downregulation of endogenous RNAs in dosage- and time-dependent manners. Furthermore, an ABA inducible split dCas13b system was generated to achieve temporally controlled deposition of m[6]A at specific sites on cellular RNAs through conditional assembly and disassembly of split dCas13b fusion proteins. We also showed that the activities of split Cas13b/dCas13b systems can be modulated by light via using a photoactivatable ABA derivative. Overall, these split Cas13b/dCas13b platforms expand the existing repertoire of the CRISPR and RNA regulation toolkit to achieve targeted manipulation of RNAs in native cellular environments with minimal functional disruption to these endogenous RNAs.

RevDate: 2023-03-06

Zhu Y, Yin S, Z Li (2023)

Mechanism of inhibition of CRISPR-Cas9 by anti-CRISPR protein AcrIIC1.

Biochemical and biophysical research communications, 654:34-39 pii:S0006-291X(23)00251-6 [Epub ahead of print].

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems are bacterial and archaeal defense mechanisms against invading phages and viruses. To overcome these defenses, phages and other mobile genetic elements (MGEs) have evolved multiple anti-CRISPR proteins (Acrs) that can inhibit the function of CRISPR-Cas systems. The AcrIIC1 protein has been shown to be able to inhibit the activity of Neisseria meningitidis Cas9 (NmeCas9) in both bacteria and human cells. Here, we solve the structure of AcrIIC1 in complex with the HNH domain of NmeCas9 using X-ray crystallography. The structure shows that AcrIIC1 binds to the catalytic sites of the HNH domain, preventing it from accessing the DNA target. In addition, our biochemical data show that AcrIIC1 is a broad-spectrum inhibitor targeting Cas9 enzymes from different subtypes. Taken together, the structure and biochemical analysis reveal the molecular mechanism of AcrIIC1-mediated Cas9 inhibition and provide new insights into regulatory tools for Cas9-based applications.

RevDate: 2023-03-08
CmpDate: 2023-03-08

Shen B, Li L, Liu C, et al (2023)

Mesoporous Nanozyme-Enhanced DNA Tetrahedron Electrochemiluminescent Biosensor with Three-Dimensional Walking Nanomotor-Mediated CRISPR/Cas12a for Ultrasensitive Detection of Exosomal microRNA.

Analytical chemistry, 95(9):4486-4495.

Exosomal microRNAs (exomiRNAs) have emerged as ideal biomarkers for early clinical diagnostics. The accurate detection of exomiRNAs plays a crucial role in facilitating clinical applications. Herein, an ultrasensitive electrochemiluminescent (ECL) biosensor was constructed using three-dimensional (3D) walking nanomotor-mediated CRISPR/Cas12a and tetrahedral DNA nanostructures (TDNs)-modified nanoemitters (TCPP-Fe@HMUiO@Au-ABEI) for exomiR-155 detection. Initially, the 3D walking nanomotor-mediated CRISPR/Cas12a strategy could effectively convert the target exomiR-155 into amplified biological signals for improving the sensitivity and specificity. Then, TCPP-Fe@HMUiO@Au nanozymes with excellent catalytic performance were used to amplify ECL signals because of the enhanced mass transfer and increased catalytic active sites, originating from its high surface areas (601.83 m[2]/g), average pore size (3.46 nm), and large pore volumes (0.52 cm[3]/g). Meanwhile, the TDNs as the scaffold to fabricate "bottom-up" anchor bioprobes could improve the trans-cleavage efficiency of Cas12a. Consequently, this biosensor achieved the limit of detection down to 273.20 aM ranging from 1.0 fM to 1.0 nM. Furthermore, the biosensor could discriminate breast cancer patients evidently by analyzing exomiR-155, and these results conformed to that of qRT-PCR. Thus, this work provides a promising tool for early clinical diagnostics.

RevDate: 2023-03-06

Bleriot I, Blasco L, Pacios O, et al (2023)

Proteomic Study of the Interactions between Phages and the Bacterial Host Klebsiella pneumoniae.

Microbiology spectrum [Epub ahead of print].

Phages and bacteria have acquired resistance mechanisms for protection. In this context, the aims of the present study were to analyze the proteins isolated from 21 novel lytic phages of Klebsiella pneumoniae in search of defense mechanisms against bacteria and also to determine the infective capacity of the phages. A proteomic study was also conducted to investigate the defense mechanisms of two clinical isolates of K. pneumoniae infected by phages. For this purpose, the 21 lytic phages were sequenced and de novo assembled. The host range was determined in a collection of 47 clinical isolates of K. pneumoniae, revealing the variable infective capacity of the phages. Genome sequencing showed that all of the phages were lytic phages belonging to the order Caudovirales. Phage sequence analysis revealed that the proteins were organized in functional modules within the genome. Although most of the proteins have unknown functions, multiple proteins were associated with defense mechanisms against bacteria, including the restriction-modification system, the toxin-antitoxin system, evasion of DNA degradation, blocking of host restriction and modification, the orphan CRISPR-Cas system, and the anti-CRISPR system. Proteomic study of the phage-host interactions (i.e., between isolates K3574 and K3320, which have intact CRISPR-Cas systems, and phages vB_KpnS-VAC35 and vB_KpnM-VAC36, respectively) revealed the presence of several defense mechanisms against phage infection (prophage, defense/virulence/resistance, oxidative stress and plasmid proteins) in the bacteria, and of the Acr candidate (anti-CRISPR protein) in the phages. IMPORTANCE Researchers, including microbiologists and infectious disease specialists, require more knowledge about the interactions between phages and their bacterial hosts and about their defense mechanisms. In this study, we analyzed the molecular mechanisms of viral and bacterial defense in phages infecting clinical isolates of K. pneumoniae. Viral defense mechanisms included restriction-modification system evasion, the toxin-antitoxin (TA) system, DNA degradation evasion, blocking of host restriction and modification, and resistance to the abortive infection system, anti-CRISPR and CRISPR-Cas systems. Regarding bacterial defense mechanisms, proteomic analysis revealed expression of proteins involved in the prophage (FtsH protease modulator), plasmid (cupin phosphomannose isomerase protein), defense/virulence/resistance (porins, efflux pumps, lipopolysaccharide, pilus elements, quorum network proteins, TA systems, and methyltransferases), oxidative stress mechanisms, and Acr candidates (anti-CRISPR protein). The findings reveal some important molecular mechanisms involved in the phage-host bacterial interactions; however, further study in this field is required to improve the efficacy of phage therapy.

RevDate: 2023-03-07

Kaltenbrunner A, Reimann V, Hoffmann UA, et al (2023)

Regulation of pSYSA defense plasmid copy number in Synechocystis through RNase E and a highly transcribed asRNA.

Frontiers in microbiology, 14:1112307.

Synthetic biology approaches toward the development of cyanobacterial producer strains require the availability of appropriate sets of plasmid vectors. A factor for the industrial usefulness of such strains is their robustness against pathogens, such as bacteriophages infecting cyanobacteria. Therefore, it is of great interest to understand the native plasmid replication systems and the CRISPR-Cas based defense mechanisms already present in cyanobacteria. In the model cyanobacterium Synechocystis sp. PCC 6803, four large and three smaller plasmids exist. The ~100 kb plasmid pSYSA is specialized in defense functions by encoding all three CRISPR-Cas systems and several toxin-antitoxin systems. The expression of genes located on pSYSA depends on the plasmid copy number in the cell. The pSYSA copy number is positively correlated with the expression level of the endoribonuclease E. As molecular basis for this correlation we identified the RNase E-mediated cleavage within the pSYSA-encoded ssr7036 transcript. Together with a cis-encoded abundant antisense RNA (asRNA1), this mechanism resembles the control of ColE1-type plasmid replication by two overlapping RNAs, RNA I and II. In the ColE1 mechanism, two non-coding RNAs interact, supported by the small protein Rop, which is encoded separately. In contrast, in pSYSA the similar-sized protein Ssr7036 is encoded within one of the interacting RNAs and it is this mRNA that likely primes pSYSA replication. Essential for plasmid replication is furthermore the downstream encoded protein Slr7037 featuring primase and helicase domains. Deletion of slr7037 led to the integration of pSYSA into the chromosome or the other large plasmid pSYSX. Moreover, the presence of slr7037 was required for successful replication of a pSYSA-derived vector in another model cyanobacterium, Synechococcus elongatus PCC 7942. Therefore, we annotated the protein encoded by slr7037 as Cyanobacterial Rep protein A1 (CyRepA1). Our findings open new perspectives on the development of shuttle vectors for genetic engineering of cyanobacteria and of modulating the activity of the entire CRISPR-Cas apparatus in Synechocystis sp. PCC 6803.

RevDate: 2023-03-07
CmpDate: 2023-03-07

Elston KM, Maeda GP, Perreau J, et al (2023)

Addressing the challenges of symbiont-mediated RNAi in aphids.

PeerJ, 11:e14961.

Because aphids are global agricultural pests and models for bacterial endosymbiosis, there is a need for reliable methods to study and control their gene function. However, current methods available for aphid gene knockout and knockdown of gene expression are often unreliable and time consuming. Techniques like CRISPR-Cas genome editing can take several months to achieve a single gene knockout because they rely on aphids going through a cycle of sexual reproduction, and aphids often lack strong, consistent levels of knockdown when fed or injected with molecules that induce an RNA interference (RNAi) response. In the hopes of addressing these challenges, we attempted to adapt a new method called symbiont-mediated RNAi (smRNAi) for use in aphids. smRNAi involves engineering a bacterial symbiont of the insect to continuously supply double-stranded RNA (dsRNA) inside the insect body. This approach has been successful in thrips, kissing bugs, and honeybees. We engineered the laboratory Escherichia coli strain HT115 and the native aphid symbiont Serratia symbiotica CWBI-2.3[T] to produce dsRNA inside the gut of the pea aphid (Acyrthosiphon pisum) targeting salivary effector protein (C002) or ecdysone receptor genes. For C002 assays, we also tested co-knockdown with an aphid nuclease (Nuc1) to reduce RNA degradation. However, we found that smRNAi was not a reliable method for aphid gene knockdown under our conditions. We were unable to consistently achieve the expected phenotypic changes with either target. However, we did see indications that elements of the RNAi pathway were modestly upregulated, and expression of some targeted genes appeared to be somewhat reduced in some trials. We conclude with a discussion of the possible avenues through which smRNAi, and aphid RNAi in general, could be improved in the future.

RevDate: 2023-03-07

Ewaisha R, KS Anderson (2023)

Immunogenicity of CRISPR therapeutics-Critical considerations for clinical translation.

Frontiers in bioengineering and biotechnology, 11:1138596.

CRISPR offers new hope for many patients and promises to transform the way we think of future therapies. Ensuring safety of CRISPR therapeutics is a top priority for clinical translation and specific recommendations have been recently released by the FDA. Rapid progress in the preclinical and clinical development of CRISPR therapeutics leverages years of experience with gene therapy successes and failures. Adverse events due to immunogenicity have been a major setback that has impacted the field of gene therapy. As several in vivo CRISPR clinical trials make progress, the challenge of immunogenicity remains a significant roadblock to the clinical availability and utility of CRISPR therapeutics. In this review, we examine what is currently known about the immunogenicity of CRISPR therapeutics and discuss several considerations to mitigate immunogenicity for the design of safe and clinically translatable CRISPR therapeutics.

RevDate: 2023-03-07
CmpDate: 2023-03-07

Zhou S, Deng L, Dong J, et al (2023)

Electrochemical detection of the p53 gene using exponential amplification reaction (EXPAR) and CRISPR/Cas12a reactions.

Mikrochimica acta, 190(4):113.

An improved electrochemical sensor has been developed for sensitive detection of the p53 gene based on exponential amplification reaction (EXPAR) and CRISPR/Cas12a. Restriction endonuclease BstNI is introduced to specifically identify and cleave the p53 gene, generating primers to trigger the EXPAR cascade amplification. A large number of amplified products are then obtained to enable the lateral cleavage activity of CRISPR/Cas12a. For electrochemical detection, the amplified product activates Cas12a to digest the designed block probe, which allows the signal probe to be captured by the reduced graphene oxide-modified electrode (GCE/RGO), resulting in an enhanced electrochemical signal. Notably, the signal probe is labeled with large amounts of methylene blue (MB). Compared with traditional endpoint decoration, the special signal probe effectively amplifies the electrochemical signals by a factor of about 15. Experimental results show that the electrochemical sensor exhibits wide ranges from 500 aM to 10 pM and 10 pM to 1 nM, as well as a relatively low limit detection of 0.39 fM, which is about an order of magnitude lower than that of fluorescence detection. Moreover, the proposed sensor shows reliable application capability in real human serum, indicating that this work has great prospects for the construction of a CRISPR-based ultra-sensitive detection platform.

RevDate: 2023-03-07
CmpDate: 2023-03-07

Wang Y, Peng Y, Li S, et al (2023)

The development of a fluorescence/colorimetric biosensor based on the cleavage activity of CRISPR-Cas12a for the detection of non-nucleic acid targets.

Journal of hazardous materials, 449:131044.

Nano-biosensors are of great significance for the analysis and detection of important biological targets. Surprisingly, the CRISPR-Cas12a system not only provides us with excellent gene editing capabilities, it also plays an important role in biosensing due to its high base resolution and high levels of sensitivity. However, most CRISPR-Cas12a-based sensors are limited by their recognition and output modes, are therefore only utilized for the detection of nucleic acids using fluorescence as an output signal. In the present study, we further explored the potential application of CRISPR-Cas12a and developed a CRISPR-Cas12a-based fluorescence/colorimetric biosensor (UCNPs-Cas12a/hydrogel-MOF-Cas12a) that provides an efficient targeting system for small molecules and protein targets. These two sensors yield multiple types of signal outputs by converting the target molecule into a deoxyribonucleic acid (DNA) signal input system using aptamers, amplifying the DNA signal by catalyzed hairpin assembly (CHA), and then combining CRISPR-Cas12a with various nanomaterials. UCNPs-Cas12a/hydrogel-MOF-Cas12a exhibited prominent sensitivity and stability for the detection of estradiol (E2) and prostate-specific antigen (PSA), and was successfully applied for the detection of these targets in milk and serum samples. A major advantage of the hydrogel-MOF-Cas12a system is that the signal output can be observed directly. When combined with aptamers and nanomaterials, CRISPR-Cas12a can be used to target multiple targets, with a diverse array of signal outputs. Our findings create a foundation for the development of CRISPR-Cas12a-based technologies for application in the fields of food safety, environmental monitoring, and clinical diagnosis.

RevDate: 2023-03-07
CmpDate: 2023-03-07

Eccleston A (2023)

CRISPR base editing protects the heart.

Nature reviews. Drug discovery, 22(3):179.

RevDate: 2023-03-07
CmpDate: 2023-03-07

Wang W, Zhou S, Cheng Z, et al (2023)

A glutathione-sensitive cationic polymer delivery of CRISPR-Cas9 RNA plasmid for targeting nasopharyngeal carcinoma gene therapy.

Colloids and surfaces. B, Biointerfaces, 223:113146.

CRISPR-Cas9 technology has been proven to be the most straightforward and accurate tool for gene therapy, but some limitations, such as the inefficient transfection or inability to precisely target, prevent the gene therapy from achieving the desired therapeutic effect. To overcome these, a kind of glutathione-sensitive cationic vectors, hyperbranched polyamide amine (HPAA) was designed for Delivery of CRISPR-Cas9 RNA plasmid, and the cyclic RGD (Arg-Gly-Asp) was conjugated for the targeted treatment of nasopharyngeal carcinoma (NPC). Disulfide bonds in HPAA segments can specifically respond to the high glutathione concentration in the tumor microenvironment. Meanwhile, RGD could especially interact to the integrin αvβ3 receptors which are highly expressed on the surface of NPC tumor cells. The results showed that more HPAA-RGD/SGK3-gRNA complexes could be uptaken by NPC HNE-1 cells after RGD was conjugated, and then the plasmid could be accumulated in the NPC tumor as well, which may assure the satisfied NPC therapy effect in vivo. In transfection assays, this complex showed the acceptable gene transfection efficiency in vitro and the obvious tumor inhibition effect in vivo, suggested a potential application in gene therapy to NPC.

RevDate: 2023-03-07
CmpDate: 2023-03-07

Yamada K, Yamamoto T, Uwasa K, et al (2023)

The establishment of multiple knockout mutants of Colletotrichum orbiculare by CRISPR-Cas9 and Cre-loxP systems.

Fungal genetics and biology : FG & B, 165:103777.

Colletotrichum orbiculare is employed as a model fungus to analyze molecular aspects of plant-fungus interactions. Although gene disruption via homologous recombination (HR) was established for C. orbiculare, this approach is laborious due to its low efficiency. Here we developed methods to generate multiple knockout mutants of C. orbiculare efficiently. We first found that CRISPR-Cas9 system massively promoted gene-targeting efficiency. By transiently introducing a CRISPR-Cas9 vector, more than 90% of obtained transformants were knockout mutants. Furthermore, we optimized a self-excision Cre-loxP marker recycling system for C. orbiculare because a limited availability of desired selective markers hampers sequential gene disruption. In this system, the integrated selective marker is removable from the genome via Cre recombinase driven by a xylose-inducible promoter, enabling the reuse of the same selective marker for the next transformation. Using our CRISPR-Cas9 and Cre-loxP systems, we attempted to identify functional sugar transporters involved in fungal virulence. Multiple disruptions of putative quinate transporter genes restricted fungal growth on media containing quinate as a sole carbon source, confirming their functionality as quinate transporters. However, our analyses showed that quinate acquisition was dispensable for infection to host plants. In addition, we successfully built mutations of 17 cellobiose transporter genes in a strain. From the data of knockout mutants that we established in this study, we inferred that repetitive rounds of gene disruption using CRISPR-Cas9 and Cre-loxP systems do not cause adverse effects on fungal virulence and growth. Therefore, these systems will be powerful tools to perform a systematic loss-of-function approach for C. orbiculare.


ESP Quick Facts

ESP Origins

In the early 1990's, Robert Robbins was a faculty member at Johns Hopkins, where he directed the informatics core of GDB — the human gene-mapping database of the international human genome project. To share papers with colleagues around the world, he set up a small paper-sharing section on his personal web page. This small project evolved into The Electronic Scholarly Publishing Project.

ESP Support

In 1995, Robbins became the VP/IT of the Fred Hutchinson Cancer Research Center in Seattle, WA. Soon after arriving in Seattle, Robbins secured funding, through the ELSI component of the US Human Genome Project, to create the original ESP.ORG web site, with the formal goal of providing free, world-wide access to the literature of classical genetics.

ESP Rationale

Although the methods of molecular biology can seem almost magical to the uninitiated, the original techniques of classical genetics are readily appreciated by one and all: cross individuals that differ in some inherited trait, collect all of the progeny, score their attributes, and propose mechanisms to explain the patterns of inheritance observed.

ESP Goal

In reading the early works of classical genetics, one is drawn, almost inexorably, into ever more complex models, until molecular explanations begin to seem both necessary and natural. At that point, the tools for understanding genome research are at hand. Assisting readers reach this point was the original goal of The Electronic Scholarly Publishing Project.

ESP Usage

Usage of the site grew rapidly and has remained high. Faculty began to use the site for their assigned readings. Other on-line publishers, ranging from The New York Times to Nature referenced ESP materials in their own publications. Nobel laureates (e.g., Joshua Lederberg) regularly used the site and even wrote to suggest changes and improvements.

ESP Content

When the site began, no journals were making their early content available in digital format. As a result, ESP was obliged to digitize classic literature before it could be made available. For many important papers — such as Mendel's original paper or the first genetic map — ESP had to produce entirely new typeset versions of the works, if they were to be available in a high-quality format.

ESP Help

Early support from the DOE component of the Human Genome Project was critically important for getting the ESP project on a firm foundation. Since that funding ended (nearly 20 years ago), the project has been operated as a purely volunteer effort. Anyone wishing to assist in these efforts should send an email to Robbins.

ESP Plans

With the development of methods for adding typeset side notes to PDF files, the ESP project now plans to add annotated versions of some classical papers to its holdings. We also plan to add new reference and pedagogical material. We have already started providing regularly updated, comprehensive bibliographies to the ESP.ORG site.

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By delivering the Cas9 nuclease, complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be precisely cut at any desired location, allowing existing genes to be removed and/or new ones added. That is, the CRISPR-Cas system provides a tool for the cut-and-paste editing of genomes. Welcome to the brave new world of genome editing. R. Robbins

Electronic Scholarly Publishing
961 Red Tail Lane
Bellingham, WA 98226

E-mail: RJR8222 @

Papers in Classical Genetics

The ESP began as an effort to share a handful of key papers from the early days of classical genetics. Now the collection has grown to include hundreds of papers, in full-text format.

Digital Books

Along with papers on classical genetics, ESP offers a collection of full-text digital books, including many works by Darwin (and even a collection of poetry — Chicago Poems by Carl Sandburg).


ESP now offers a much improved and expanded collection of timelines, designed to give the user choice over subject matter and dates.


Biographical information about many key scientists.

Selected Bibliographies

Bibliographies on several topics of potential interest to the ESP community are now being automatically maintained and generated on the ESP site.

ESP Picks from Around the Web (updated 07 JUL 2018 )