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Bibliography on: CRISPR-Cas

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ESP: PubMed Auto Bibliography 22 Jan 2022 at 01:32 Created: 

CRISPR-Cas

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®)

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RevDate: 2022-01-20

Klompe SE, Jaber N, Beh LY, et al (2022)

Evolutionary and mechanistic diversity of Type I-F CRISPR-associated transposons.

Molecular cell pii:S1097-2765(21)01083-2 [Epub ahead of print].

Canonical CRISPR-Cas systems utilize RNA-guided nucleases for targeted cleavage of foreign nucleic acids, whereas some nuclease-deficient CRISPR-Cas complexes have been repurposed to direct the insertion of Tn7-like transposons. Here, we established a bioinformatic and experimental pipeline to comprehensively explore the diversity of Type I-F CRISPR-associated transposons. We report DNA integration for 20 systems and identify a highly active subset that exhibits complete orthogonality in transposon DNA mobilization. We reveal the modular nature of CRISPR-associated transposons by exploring the horizontal acquisition of targeting modules and by characterizing a system that encodes both a programmable, RNA-dependent pathway, and a fixed, RNA-independent pathway. Finally, we analyzed transposon-encoded cargo genes and found the striking presence of anti-phage defense systems, suggesting a role in transmitting innate immunity between bacteria. Collectively, this study substantially advances our biological understanding of CRISPR-associated transposon function and expands the suite of RNA-guided transposases for programmable, large-scale genome engineering.

RevDate: 2022-01-20

Tan R, Krueger RK, Gramelspacher MJ, et al (2022)

Cas11 enables genome engineering in human cells with compact CRISPR-Cas3 systems.

Molecular cell pii:S1097-2765(21)01137-0 [Epub ahead of print].

Leading CRISPR-Cas technologies employ Cas9 and Cas12 enzymes that generate RNA-guided dsDNA breaks. Yet, the most abundant microbial adaptive immune systems, Type I CRISPRs, are under-exploited for eukaryotic applications. Here, we report the adoption of a minimal CRISPR-Cas3 from Neisseria lactamica (Nla) type I-C system to create targeted large deletions in the human genome. RNP delivery of its processive Cas3 nuclease and target recognition complex Cascade can confer ∼95% editing efficiency. Unexpectedly, NlaCascade assembly in bacteria requires internal translation of a hidden component Cas11 from within the cas8 gene. Furthermore, expressing a separately encoded NlaCas11 is the key to enable plasmid- and mRNA-based editing in human cells. Finally, we demonstrate that supplying cas11 is a universal strategy to systematically implement divergent I-C, I-D, and I-B CRISPR-Cas3 editors with compact sizes, distinct PAM preferences, and guide orthogonality. These findings greatly expand our ability to engineer long-range genome edits.

RevDate: 2022-01-20

Zhang Q, Zhao S, Tian X, et al (2022)

Development of a CRISPR-Cas-Based Biosensor for Rapid and Sensitive Detection of 8-Oxoguanine DNA Glycosylase.

Analytical chemistry [Epub ahead of print].

8-Oxoguanine DNA glycosylase is essential for maintaining genomic integrity and stability, while its abnormal activity may lead to the disturbance in the normal DNA damage repair and the occurrence of carcinogenicity and teratogenicity. Herein, we construct a CRISPR-Cas-based biosensor for rapid and sensitive measurement of 8-oxoguanine DNA glycosylases. This biosensor involves a hairpin probe and integrates quadratic strand displacement amplification (SDA) with a CRISPR/Cas12a effector with the characteristics of rapidity (within 40 min) and isothermal assay. The presence of 8-oxoguanine DNA glycosylase can initiate the quadratic SDA to produce large amounts of activators with the assistance of polynucleotide kinase (PNK). Subsequently, the activators can bind with crRNA to activate Cas12a, cleaving signal probes and recovering Cy5 fluorescence, which can be accurately quantified by single-molecule imaging. Notably, the designed hairpin probes can effectively block the hybridization of the generated activators with free hairpin probes, endowing this biosensor with high sensitivity. In addition, the utilization of PNK instead of apurinic/apyrimidinic endonuclease (APE1) greatly simplifies the experimental procedure to only a one-step reaction. The introduction of a single-molecule detection further reduces the sample consumption and improves the sensitivity. This biosensor displays a detection limit of 4.24 × 10-9 U μL-1, and it can accurately quantify cellular human 8-oxoguanine DNA glycosylase at a single-cell level. Furthermore, this biosensor can be applied for the screening of inhibitors, the analysis of kinetic parameters, and the discrimination of cancer cells from normal cells, with potential applications in molecular diagnostic and point-of-care testing.

RevDate: 2022-01-20

Katzmann JL, Cupido AJ, U Laufs (2022)

Gene Therapy Targeting PCSK9.

Metabolites, 12(1): pii:metabo12010070.

The last decades of research in cardiovascular prevention have been characterized by successful bench-to-bedside developments for the treatment of low-density lipoprotein (LDL) hypercholesterolemia. Recent examples include the inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) with monoclonal antibodies, small interfering RNA and antisense RNA drugs. The cumulative effects of LDL cholesterol on atherosclerosis make early, potent, and long-term reductions in LDL cholesterol desirable-ideally without the need of regular intake or application of medication and importantly, without side effects. Current reports show durable LDL cholesterol reductions in primates following one single treatment with PCSK9 gene or base editors. Use of the CRISPR/Cas system enables precise genome editing down to single-nucleotide changes. Provided safety and documentation of a reduction in cardiovascular events, this novel technique has the potential to fundamentally change our current concepts of cardiovascular prevention. In this review, the application of the CRISPR/Cas system is explained and the current state of in vivo approaches of PCSK9 editing is presented.

RevDate: 2022-01-20

Paramasivam K, Shen Y, Yuan J, et al (2022)

Advances in the Development of Phage-Based Probes for Detection of Bio-Species.

Biosensors, 12(1): pii:bios12010030.

Bacteriophages, abbreviated as "phages", have been developed as emerging nanoprobes for the detection of a wide variety of biological species, such as biomarker molecules and pathogens. Nanosized phages can display a certain length of exogenous peptides of arbitrary sequence or single-chain variable fragments (scFv) of antibodies that specifically bind to the targets of interest, such as animal cells, bacteria, viruses, and protein molecules. Metal nanoparticles generally have unique plasmon resonance effects. Metal nanoparticles such as gold, silver, and magnetism are widely used in the field of visual detection. A phage can be assembled with metal nanoparticles to form an organic-inorganic hybrid probe due to its nanometer-scale size and excellent modifiability. Due to the unique plasmon resonance effect of this composite probe, this technology can be used to visually detect objects of interest under a dark-field microscope. In summary, this review summarizes the recent advances in the development of phage-based probes for ultra-sensitive detection of various bio-species, outlining the advantages and limitations of detection technology of phage-based assays, and highlighting the commonly used editing technologies of phage genomes such as homologous recombination and clustered regularly interspaced palindromic repeats/CRISPR-associated proteins system (CRISPR-Cas). Finally, we discuss the possible scenarios for clinical application of phage-probe-based detection methods.

RevDate: 2022-01-20

Allen D, Weiss LE, Saguy A, et al (2022)

High-throughput Imaging of CRISPR- and Recombinant Adeno-associated Virus-induced DNA Damage Response in Human Hematopoietic Stem and Progenitor Cells.

The CRISPR journal [Epub ahead of print].

CRISPR-Cas technology has revolutionized gene editing, but concerns remain due to its propensity for off-target interactions. This, combined with genotoxicity related to both CRISPR-Cas9-induced double-strand breaks and transgene delivery, poses a significant liability for clinical genome-editing applications. Current best practice is to optimize genome-editing parameters in preclinical studies. However, quantitative tools that measure off-target interactions and genotoxicity are costly and time-consuming, limiting the practicality of screening large numbers of potential genome-editing reagents and conditions. Here, we show that flow-based imaging facilitates DNA damage characterization of hundreds of human hematopoietic stem and progenitor cells per minute after treatment with CRISPR-Cas9 and recombinant adeno-associated virus serotype 6. With our web-based platform that leverages deep learning for image analysis, we find that greater DNA damage response is observed for guide RNAs with higher genome-editing activity, differentiating even single on-target guide RNAs with different levels of off-target interactions. This work simplifies the characterization and screening process of genome-editing parameters toward enabling safer and more effective gene-therapy applications.

RevDate: 2022-01-20

Aviram N, Thornal AN, Zeevi D, et al (2022)

Different modes of spacer acquisition by the Staphylococcus epidermidis type III-A CRISPR-Cas system.

Nucleic acids research pii:6511979 [Epub ahead of print].

CRISPR-Cas systems provide prokaryotic organisms with an adaptive defense mechanism that acquires immunological memories of infections. This is accomplished by integration of short fragments from the genome of invaders such as phages and plasmids, called 'spacers', into the CRISPR locus of the host. Depending on their genetic composition, CRISPR-Cas systems can be classified into six types, I-VI, however spacer acquisition has been extensively studied only in type I and II systems. Here, we used an inducible spacer acquisition assay to study this process in the type III-A CRISPR-Cas system of Staphylococcus epidermidis, in the absence of phage selection. Similarly to type I and II spacer acquisition, this type III system uses Cas1 and Cas2 to preferentially integrate spacers from the chromosomal terminus and free dsDNA ends produced after DNA breaks, in a manner that is enhanced by the AddAB DNA repair complex. Surprisingly, a different mode of spacer acquisition from rRNA and tRNA loci, which spans only the transcribed sequences of these genes and is not enhanced by AddAB, was also detected. Therefore, our findings reveal both common mechanistic principles that may be conserved in all CRISPR-Cas systems, as well as unique and intriguing features of type III spacer acquisition.

RevDate: 2022-01-20
CmpDate: 2022-01-20

Kim HJ, Park JM, Lee S, et al (2022)

Efficient CRISPR-Cas9-based knockdown of RUNX2 to induce chondrogenic differentiation of stem cells.

Biomaterials science, 10(2):514-523.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system recognizes and deletes specific nucleotide sequences in cells for gene editing. This study aimed to edit and knockdown the RUNX2 gene, a key transcription factor that is directly involved in all stages of stem cell differentiation into osteoblasts. The RUNX2 gene was depleted using the CRISPR-Cas9 system to inhibit osteoblast differentiation of stem cells. shRNA vectors targeting RUNX2 were used as a control. The surface of nanoparticles (NPs) was coated with the cationic polymer linear polyethyleneimine. Thereafter, negatively charged CRISPR-Cas9 and shRNA vectors were complexed with positively charged NPs via ionic interactions. Several analytical methods were used to determine the size, surface charge, and morphology of NPs and to characterize the complexed genes. NPs complexed with CRISPR-Cas9 and shRNA vectors were delivered into human mesenchymal stem cells (hMSCs) via endocytosis. The mRNA and protein expression patterns of various genes in hMSCs were measured over time following internalization of NPs complexed with CRISPR-Cas9 and shRNA vectors in two- and three-dimensional culture systems. Knockdown of the RUNX2 gene decreased osteogenic differentiation and increased chondrogenic differentiation of hMSCs. As a result of investigating the efficiency of NPs complexed with CRISPR-Cas9 (CASP-NPs), Runx2 effectively knocked down in mesenchymal stem cells to enhance differentiation into chondrocytes, therefore CASP-NPs proved to be an effective gene carrier in hMSCs.

RevDate: 2022-01-20
CmpDate: 2022-01-20

Kowalczyk JE, Saha S, MR Mäkelä (2021)

Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus Dichomitus squalens.

Biomolecules, 11(10):.

Dichomitus squalens is an emerging reference species that can be used to investigate white-rot fungal plant biomass degradation, as it has flexible physiology to utilize different types of biomass as sources of carbon and energy. Recent comparative (post-) genomic studies on D. squalens resulted in an increasingly detailed knowledge of the genes and enzymes involved in the lignocellulose breakdown in this fungus and showed a complex transcriptional response in the presence of lignocellulose-derived compounds. To fully utilize this increasing amount of data, efficient and reliable genetic manipulation tools are needed, e.g., to characterize the function of certain proteins in vivo and facilitate the construction of strains with enhanced lignocellulolytic capabilities. However, precise genome alterations are often very difficult in wild-type basidiomycetes partially due to extremely low frequencies of homology directed recombination (HDR) and limited availability of selectable markers. To overcome these obstacles, we assessed various Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) -based strategies for selectable homology and non-homologous end joining (NHEJ) -based gene editing in D. squalens. We also showed an induction of HDR-based genetic modifications by using single-stranded oligodeoxynucleotides (ssODNs) in a basidiomycete fungus for the first time. This paper provides directions for the application of targeted CRISPR/Cas9-based genome editing in D. squalens and other wild-type (basidiomycete) fungi.

RevDate: 2022-01-20
CmpDate: 2022-01-20

Wang Y, Li J, Li S, et al (2021)

LAMP-CRISPR-Cas12-based diagnostic platform for detection of Mycobacterium tuberculosis complex using real-time fluorescence or lateral flow test.

Mikrochimica acta, 188(10):347.

A CRISPR-based nucleic acid detection platform, termed LACD (loop-mediated isothermal amplification coupled with CRISPR-Cas12a-mediated diagnostic) has been developed. In the LACD system, the core primer used in conventional LAMP (forward inner primer or backward inner primer) was engineered to contain a PAM (protospacer adjacent motif) site (TTTT) at the linker region. As a result, the LAMP amplicons contained a specific PAM site for CRISPR-Cas12a recognition. At the CRISPR-mediated detection stage, the resulting LAMP products can activate the corresponding CRISPR-Cas12a effector upon the formation of the CRISPR-Cas12a/gRNA/target DNA complex. The single-strand DNA (ssDNA) reporter molecules are then rapidly cleaved due to the CRISPR-Cas12a's trans-enzyme activity. The ssDNA degradation can then be visualized on a lateral flow biosensor or measured by a real-time fluorescence instrument. Our LACD assay allows any target sequence to be detected (even targets which do not contain any PAM sites) as long as they met the design requirement for LAMP. The feasibility of the LACD methodology for nucleic acid detection was validated on the Mycobacterium tuberculosis complex (MTC). This proof-of-concept assay can be reconfigured to detect a variety of target sequences by redesigning the engineered LAMP primers.

RevDate: 2022-01-20
CmpDate: 2022-01-20

Perez M, Angers B, Young CR, et al (2021)

Shining light on a deep-sea bacterial symbiont population structure with CRISPR.

Microbial genomics, 7(8):.

Many foundation species in chemosynthesis-based ecosystems rely on environmentally acquired symbiotic bacteria for their survival. Hence, understanding the biogeographic distributions of these symbionts at regional scales is key to understanding patterns of connectivity and predicting resilience of their host populations (and thus whole communities). However, such assessments are challenging because they necessitate measuring bacterial genetic diversity at fine resolutions. For this purpose, the recently discovered clustered regularly interspaced short palindromic repeats (CRISPR) constitutes a promising new genetic marker. These DNA sequences harboured by about half of bacteria hold their viral immune memory, and as such, might allow discrimination of different lineages or strains of otherwise indistinguishable bacteria. In this study, we assessed the potential of CRISPR as a hypervariable phylogenetic marker in the context of a population genetic study of an uncultured bacterial species. We used high-throughput CRISPR-based typing along with multi-locus sequence analysis (MLSA) to characterize the regional population structure of the obligate but environmentally acquired symbiont species Candidatus Endoriftia persephone on the Juan de Fuca Ridge. Mixed symbiont populations of Ca. Endoriftia persephone were sampled across individual Ridgeia piscesae hosts from contrasting habitats in order to determine if environmental conditions rather than barriers to connectivity are more important drivers of symbiont diversity. We showed that CRISPR revealed a much higher symbiont genetic diversity than the other housekeeping genes. Several lines of evidence imply this diversity is indicative of environmental strains. Finally, we found with both CRISPR and gene markers that local symbiont populations are strongly differentiated across sites known to be isolated by deep-sea circulation patterns. This research showed the high power of CRISPR to resolve the genetic structure of uncultured bacterial populations and represents a step towards making keystone microbial species an integral part of conservation policies for upcoming mining operations on the seafloor.

RevDate: 2022-01-20
CmpDate: 2022-01-20

Fang H, Bygrave AM, Roth RH, et al (2021)

An optimized CRISPR/Cas9 approach for precise genome editing in neurons.

eLife, 10:.

The efficient knock-in of large DNA fragments to label endogenous proteins remains especially challenging in non-dividing cells such as neurons. We developed Targeted Knock-In with Two (TKIT) guides as a novel CRISPR/Cas9 based approach for efficient, and precise, genomic knock-in. Through targeting non-coding regions TKIT is resistant to INDEL mutations. We demonstrate TKIT labeling of endogenous synaptic proteins with various tags, with efficiencies up to 42% in mouse primary cultured neurons. Utilizing in utero electroporation or viral injections in mice TKIT can label AMPAR subunits with Super Ecliptic pHluorin, enabling visualization of endogenous AMPARs in vivo using two-photon microscopy. We further use TKIT to assess the mobility of endogenous AMPARs using fluorescence recovery after photobleaching. Finally, we show that TKIT can be used to tag AMPARs in rat neurons, demonstrating precise genome editing in another model organism and highlighting the broad potential of TKIT as a method to visualize endogenous proteins.

RevDate: 2022-01-19

Fan W, Han P, Feng Q, et al (2022)

Nucleic Acid Substrate-Independent DNA Polymerization on the Exosome Membrane: A Mechanism Study and Application in Exosome Analysis.

Analytical chemistry [Epub ahead of print].

As generally acknowledged, terminal deoxynucleotidyl transferase (TdT) can only elongate DNA substrates from their 3'-OH ends. Herein, for the first time, we report that TdT-catalyzed DNA polymerization can directly proceed on the exosome membrane without the mediation of any nucleic acids. We prove that both the glycosyl and phenolic hydroxyl groups on the membrane proteins can initiate the DNA polymerization. Accordingly, we have developed powerful strategies for high-sensitive exosome profiling based on a conventional flow cytometer and an emerging CRISPR/Cas system. By using our strategy, the featured membrane protein distributions of different cancer cell-derived exosomes can be figured out, which can clearly distinguish plasma samples of breast cancer patients from those of healthy people. This work paves new ways for exosome profiling and liquid biopsy and expands the understanding of TdT, holding great significance in developing TdT-based sensing systems as well as establishing protein/nucleic acid hybrid biomaterials.

RevDate: 2022-01-19

Shi J, L Wei (2022)

Rho Kinases in Embryonic Development and Stem Cell Research.

Archivum immunologiae et therapiae experimentalis, 70(1):4.

The Rho-associated coiled-coil containing kinases (ROCKs or Rho kinases) belong to the AGC (PKA/PKG/PKC) family of serine/threonine kinases and are major downstream effectors of small GTPase RhoA, a key regulator of actin-cytoskeleton reorganization. The ROCK family contains two members, ROCK1 and ROCK2, which share 65% overall identity and 92% identity in kinase domain. ROCK1 and ROCK2 were assumed to be functionally redundant, based largely on their major common activators, their high degree kinase domain homology, and study results from overexpression with kinase constructs or chemical inhibitors. ROCK signaling research has expanded to all areas of biology and medicine since its discovery in 1996. The rapid advance is befitting ROCK's versatile functions in modulating various cell behavior, such as contraction, adhesion, migration, proliferation, polarity, cytokinesis, and differentiation. The rapid advance is noticeably driven by an extensive linking with clinical medicine, including cardiovascular abnormalities, aberrant immune responsive, and cancer development and metastasis. The rapid advance during the past decade is further powered by novel biotechnologies including CRISPR-Cas and single cell omics. Current consensus, derived mainly from gene targeting and RNA interference approaches, is that the two ROCK isoforms have overlapping and distinct cellular, physiological and pathophysiology roles. In this review, we present an overview of the milestone discoveries in ROCK research. We then focus on the current understanding of ROCK signaling in embryonic development, current research status using knockout and knockin mouse models, and stem cell research.

RevDate: 2022-01-19
CmpDate: 2022-01-19

Toma L, Barbălată T, Sanda GM, et al (2021)

CRISPR/dCas9 Transcriptional Activation of Endogenous Apolipoprotein AI and Paraoxonase 1 in Enterocytes Alleviates Endothelial Cell Dysfunction.

Biomolecules, 11(12):.

Atherosclerosis is the main cause of cardiovascular diseases with high prevalence worldwide. A promising therapeutic strategy to reverse atherosclerotic process is to improve the athero-protective potential of high-density lipoproteins (HDL). Since the small intestine is a source of HDL, we aimed to activate transcription of the endogenous HDL major proteins, apolipoprotein AI (ApoAI) and paraoxonase 1 (PON1), in enterocytes, and to evaluate their potential to correct the pro-inflammatory status of endothelial cells (EC). Caco-2 enterocytes were transfected with CRISPR activation plasmids targeting ApoAI or PON1, and their gene and protein expression were measured in cells and conditioned medium (CM). ATP binding cassette A1 and G8 transporters (ABCA1, ABCG8), scavenger receptor BI (SR-BI), and transcription regulators peroxisome proliferator-activated receptor γ (PPARγ), liver X receptors (LXRs), and sirtuin-1 (SIRT1) were assessed. Anti-inflammatory effects of CM from transfected enterocytes were estimated through its ability to inhibit tumor necrosis factor α (TNFα) activation of EC. Transcriptional activation of ApoAI or PON1 in enterocytes induces: (i) increase of their gene and protein expression, and secretion in CM; (ii) stimulation of ABCA1/G8 and SR-BI; (iii) upregulation of PPARγ, LXRs, and SIRT1. CM from transfected enterocytes attenuated the TNFα-induced inflammatory and oxidative stress in EC, by decreasing TNF receptor 1, monocyte chemoattractant protein-1, and p22phox. In conclusion, transcriptional activation of endogenous ApoAI or PON1 in enterocytes by CRISPR/dCas9 system is a realistic approach to stimulate biogenesis and function of major HDL proteins which can regulate cholesterol efflux transporters and reduce the inflammatory stress in activated EC.

RevDate: 2022-01-19
CmpDate: 2022-01-19

Johansen KH (2022)

How CRISPR/Cas9 Gene Editing Is Revolutionizing T Cell Research.

DNA and cell biology, 41(1):53-57.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 allows for precise gene targeting in mammalian cells, including T cells, allowing scientists to disrupt or edit specific genes of interest. This has enabled immunologists to investigate T cell functions as well as opened the path for novel therapeutics involving gene editing of T cells ex vivo before transferring these back to patients to increase T cell efficacy. This review outlines how CRISPR/Cas9 has transformed T cell research allowing immunologists to rapidly probe the roles of genes in T cells thus paving the way for novel therapeutics. Furthermore, this review describes how these tools reduce the requirement for genetic mouse models, while increasing the translational potential of T cell research.

RevDate: 2022-01-19
CmpDate: 2022-01-19

Kumari P, Sturgeon M, Bonde G, et al (2022)

Generating Zebrafish RNA-Less Mutant Alleles by Deleting Gene Promoters with CRISPR/Cas9.

Methods in molecular biology (Clifton, N.J.), 2403:91-106.

Danio rerio (zebrafish), traditionally used in forward genetic screens, has in the last decade become a popular model for reverse genetic studies with the introduction of TALENS, zinc finger nucleases, and CRISPR/Cas9. Unexpectedly, homozygous frameshift mutations generated by these tools frequently result in phenotypes that are less penetrant than those seen in embryos injected with antisense morpholino oligonucleotides targeting the same gene. One explanation for the difference is that some frameshift mutations result in nonsense-mediated decay of the gene transcript, a process which can induce expression of homologous genes. This form of genetic compensation, called transcriptional adaptation, does not occur when the mutant allele results in no RNA transcripts being produced from the targeted gene. Such RNA-less mutants can be generated by deleting a gene's promoter using a pair of guide RNAs and Cas9 protein. Here, we present a protocol and use it to generate alleles of arhgap29b and slc41a1 that lack detectable zygotic transcription. In the case of the arhgap29b mutant, an emerging phenotype did not segregate with the promoter deletion mutation, highlighting the potential for off-target mutagenesis with these tools. In summary, this chapter describes a method to generate zebrafish mutants that avoid a form of genetic compensation that occurs in many frameshift mutants.

RevDate: 2022-01-19
CmpDate: 2022-01-19

Xu K, Y Li (2022)

Developing Rice Mutants Using CRISPR/Cas9-Based Genome Editing Technology.

Methods in molecular biology (Clifton, N.J.), 2400:11-19.

Genome editing provides advanced techniques that enable the precise and efficient targeted modification of an organism's genome. It is a powerful tool for understanding gene functions and developing valuable new traits in crops such as rice. The clustered regularly interspersed short palindromic repeats (CRISPR)/Cas9 system has recently emerged as an alternative nuclease-based method for efficient and versatile genome editing. In the CRISPR/Cas9 system, only 20 nt within the single guide RNA (sgRNA) needs to be changed. The convenience of operating and few limitations on target loci make the CRISPR/Cas9 system an ideal tool in our research. Here, we describe a detailed protocol of the CRISPR/Cas9 system to generate rice mutants used in our recent gene function studies.

RevDate: 2022-01-19
CmpDate: 2022-01-19

Basak I, Hansen RA, Ward ME, et al (2021)

Deficiency of the Lysosomal Protein CLN5 Alters Lysosomal Function and Movement.

Biomolecules, 11(10):.

Batten disease is a devastating, childhood, rare neurodegenerative disease characterised by the rapid deterioration of cognition and movement, leading to death within ten to thirty years of age. One of the thirteen Batten disease forms, CLN5 Batten disease, is caused by mutations in the CLN5 gene, leading to motor deficits, mental deterioration, cognitive impairment, visual impairment, and epileptic seizures in children. A characteristic pathology in CLN5 Batten disease is the defects in lysosomes, leading to neuronal dysfunction. In this study, we aimed to investigate the lysosomal changes in CLN5-deficient human neurons. We used an induced pluripotent stem cell system, which generates pure human cortical-like glutamatergic neurons. Using CRISPRi, we inhibited the expression of CLN5 in human neurons. The CLN5-deficient human neurons showed reduced acidic organelles and reduced lysosomal enzyme activity measured by microscopy and flow cytometry. Furthermore, the CLN5-deficient human neurons also showed impaired lysosomal movement-a phenotype that has never been reported in CLN5 Batten disease. Lysosomal trafficking is key to maintain local degradation of cellular wastes, especially in long neuronal projections, and our results from the human neuronal model present a key finding to understand the underlying lysosomal pathology in neurodegenerative diseases.

RevDate: 2022-01-19
CmpDate: 2022-01-19

Goullée H, Taylor RL, Forrest ARR, et al (2021)

Improved CRISPR/Cas9 gene editing in primary human myoblasts using low confluency cultures on Matrigel.

Skeletal muscle, 11(1):23.

BACKGROUND: CRISPR/Cas9 is an invaluable tool for studying cell biology and the development of molecular therapies. However, delivery of CRISPR/Cas9 components into some cell types remains a major hurdle. Primary human myoblasts are a valuable cell model for muscle studies, but are notoriously difficult to transfect. There are currently no commercial lipofection protocols tailored for primary myoblasts, and most generic guidelines simply recommend transfecting healthy cells at high confluency. This study aimed to maximize CRISPR/Cas9 transfection and editing in primary human myoblasts.

METHODS: Since increased cell proliferation is associated with increased transfection efficiency, we investigated two factors known to influence myoblast proliferation: cell confluency, and a basement membrane matrix, Matrigel. CRISPR/Cas9 editing was performed by delivering Cas9 ribonucleoprotein complexes via lipofection into primary human myoblasts, cultured in wells with or without a Matrigel coating, at low (~ 40%) or high (~ 80%) confluency.

RESULTS: Cells transfected at low confluency on Matrigel-coated wells had the highest levels of transfection, and were most effectively edited across three different target loci, achieving a maximum editing efficiency of 93.8%. On average, editing under these conditions was >4-fold higher compared to commercial recommendations (high confluency, uncoated wells).

CONCLUSION: This study presents a simple, effective and economical method of maximizing CRISPR/Cas9-mediated gene editing in primary human myoblasts. This protocol could be a valuable tool for improving the genetic manipulation of cultured human skeletal muscle cells, and potentially be adapted for use in other cell types.

RevDate: 2022-01-19
CmpDate: 2022-01-19

Wen W, Quan ZJ, Li SA, et al (2021)

Effective control of large deletions after double-strand breaks by homology-directed repair and dsODN insertion.

Genome biology, 22(1):236.

BACKGROUND: After repairing double-strand breaks (DSBs) caused by CRISPR-Cas9 cleavage, genomic damage, such as large deletions, may have pathogenic consequences.

RESULTS: We show that large deletions are ubiquitous but are dependent on editing sites and cell types. Human primary T cells display more significant deletions than hematopoietic stem and progenitor cells (HSPCs), whereas we observe low levels in induced pluripotent stem cells (iPSCs). We find that the homology-directed repair (HDR) with single-stranded oligodeoxynucleotides (ssODNs) carrying short homology reduces the deletion damage by almost half, while adeno-associated virus (AAV) donors with long homology reduce large deletions by approximately 80%. In the absence of HDR, the insertion of a short double-stranded ODN by NHEJ reduces deletion indexes by about 60%.

CONCLUSIONS: Timely bridging of broken ends by HDR and NHEJ vastly decreases the unintended consequences of dsDNA cleavage. These strategies can be harnessed in gene editing applications to attenuate unintended outcomes.

RevDate: 2022-01-19
CmpDate: 2022-01-19

Li Y, Chen J, Tsai SQ, et al (2021)

Easy-Prime: a machine learning-based prime editor design tool.

Genome biology, 22(1):235.

Prime editing is a revolutionary genome-editing technology that can make a wide range of precise edits in DNA. However, designing highly efficient prime editors (PEs) remains challenging. We develop Easy-Prime, a machine learning-based program trained with multiple published data sources. Easy-Prime captures both known and novel features, such as RNA folding structure, and optimizes feature combinations to improve editing efficiency. We provide optimized PE design for installation of 89.5% of 152,351 GWAS variants. Easy-Prime is available both as a command line tool and an interactive PE design server at: http://easy-prime.cc/ .

RevDate: 2022-01-19
CmpDate: 2022-01-19

Fix SM, Jazaeri AA, P Hwu (2021)

Applications of CRISPR Genome Editing to Advance the Next Generation of Adoptive Cell Therapies for Cancer.

Cancer discovery, 11(3):560-574.

Adoptive cell therapy (ACT) for cancer shows tremendous potential; however, several challenges preclude its widespread use. These include poor T-cell function in hostile tumor microenvironments, a lack of tumor-specific target antigens, and the high cost and poor scalability of cell therapy manufacturing. Creative genome-editing strategies are beginning to emerge to address each of these limitations, which has initiated the next generation of cell therapy products now entering clinical trials. CRISPR is at the forefront of this revolution, offering a simple and versatile platform for genetic engineering. This review provides a comprehensive overview of CRISPR applications that have advanced ACT. SIGNIFICANCE: The clinical impact of ACT for cancer can be expanded by implementing specific genetic modifications that enhance the potency, safety, and scalability of cellular products. Here we provide a detailed description of such genetic modifications, highlighting avenues to enhance the therapeutic efficacy and accessibility of ACT for cancer. Furthermore, we review high-throughput CRISPR genetic screens that have unveiled novel targets for cell therapy enhancement.

RevDate: 2022-01-19
CmpDate: 2022-01-19

Kim E, T Hart (2021)

Improved analysis of CRISPR fitness screens and reduced off-target effects with the BAGEL2 gene essentiality classifier.

Genome medicine, 13(1):2.

BACKGROUND: Identifying essential genes in genome-wide loss-of-function screens is a critical step in functional genomics and cancer target finding. We previously described the Bayesian Analysis of Gene Essentiality (BAGEL) algorithm for accurate classification of gene essentiality from short hairpin RNA and CRISPR/Cas9 genome-wide genetic screens.

RESULTS: We introduce an updated version, BAGEL2, which employs an improved model that offers a greater dynamic range of Bayes Factors, enabling detection of tumor suppressor genes; a multi-target correction that reduces false positives from off-target CRISPR guide RNA; and the implementation of a cross-validation strategy that improves performance ~ 10× over the prior bootstrap resampling approach. We also describe a metric for screen quality at the replicate level and demonstrate how different algorithms handle lower quality data in substantially different ways.

CONCLUSIONS: BAGEL2 substantially improves the sensitivity, specificity, and performance over BAGEL and establishes the new state of the art in the analysis of CRISPR knockout fitness screens. BAGEL2 is written in Python 3 and source code, along with all supporting files, are available on github (https://github.com/hart-lab/bagel).

RevDate: 2022-01-19
CmpDate: 2022-01-19

Markiewicz Ł, Uśpieński T, Baran B, et al (2021)

Xpo7 negatively regulates Hedgehog signaling by exporting Gli2 from the nucleus.

Cellular signalling, 80:109907.

Dynamic bidirectional transport between the nucleus and the cytoplasm is critical for the regulation of many transcription factors, whose levels inside the nucleus must be tightly controlled. Efficient shuttling across the nuclear membrane is especially crucial with regard to the Hedgehog (Hh) pathway, where the transcriptional signal depends on the fine balance between the amounts of Gli protein activator and repressor forms in the nucleus. The nuclear export machinery prevents the unchecked nuclear accumulation of Gli proteins, but the mechanistic insight into this process is limited. We show that the atypical exportin Xpo7 functions as a major nuclear export receptor that actively excludes Gli2 from the nucleus and controls the outcome of Hh signaling. We show that Xpo7 interacts with several domains of Gli2 and that this interaction is modulated by SuFu, a key negative regulator of Hh signaling. Our data pave the way for a more complete understanding of the nuclear shuttling of Gli proteins and the regulation of their transcriptional activity.

RevDate: 2022-01-19
CmpDate: 2022-01-19

Upadhyay R, Boiarsky JA, Pantsulaia G, et al (2021)

A Critical Role for Fas-Mediated Off-Target Tumor Killing in T-cell Immunotherapy.

Cancer discovery, 11(3):599-613.

T cell-based therapies have induced cancer remissions, though most tumors ultimately progress, reflecting inherent or acquired resistance including antigen escape. Better understanding of how T cells eliminate tumors will help decipher resistance mechanisms. We used a CRISPR/Cas9 screen and identified a necessary role for Fas-FasL in antigen-specific T-cell killing. We also found that Fas-FasL mediated off-target "bystander" killing of antigen-negative tumor cells. This localized bystander cytotoxicity enhanced clearance of antigen-heterogeneous tumors in vivo, a finding that has not been shown previously. Fas-mediated on-target and bystander killing was reproduced in chimeric antigen receptor (CAR-T) and bispecific antibody T-cell models and was augmented by inhibiting regulators of Fas signaling. Tumoral FAS expression alone predicted survival of CAR-T-treated patients in a large clinical trial (NCT02348216). These data suggest strategies to prevent immune escape by targeting both the antigen expression of most tumor cells and the geography of antigen-loss variants. SIGNIFICANCE: This study demonstrates the first report of in vivo Fas-dependent bystander killing of antigen-negative tumors by T cells, a phenomenon that may be contributing to the high response rates of antigen-directed immunotherapies despite tumoral heterogeneity. Small molecules that target the Fas pathway may potentiate this mechanism to prevent cancer relapse.This article is highlighted in the In This Issue feature, p. 521.

RevDate: 2022-01-19
CmpDate: 2022-01-19

Li J, Yuan S, Norgard RJ, et al (2021)

Epigenetic and Transcriptional Control of the Epidermal Growth Factor Receptor Regulates the Tumor Immune Microenvironment in Pancreatic Cancer.

Cancer discovery, 11(3):736-753.

Although immunotherapy has revolutionized cancer care, patients with pancreatic ductal adenocarcinoma (PDA) rarely respond to these treatments, a failure that is attributed to poor infiltration and activation of T cells in the tumor microenvironment (TME). We performed an in vivo CRISPR screen and identified lysine demethylase 3A (KDM3A) as a potent epigenetic regulator of immunotherapy response in PDA. Mechanistically, KDM3A acts through Krueppel-like factor 5 (KLF5) and SMAD family member 4 (SMAD4) to regulate the expression of the epidermal growth factor receptor (EGFR). Ablation of KDM3A, KLF5, SMAD4, or EGFR in tumor cells altered the immune TME and sensitized tumors to combination immunotherapy, whereas treatment of established tumors with an EGFR inhibitor, erlotinib, prompted a dose-dependent increase in intratumoral T cells. This study defines an epigenetic-transcriptional mechanism by which tumor cells modulate their immune microenvironment and highlights the potential of EGFR inhibitors as immunotherapy sensitizers in PDA. SIGNIFICANCE: PDA remains refractory to immunotherapies. Here, we performed an in vivo CRISPR screen and identified an epigenetic-transcriptional network that regulates antitumor immunity by converging on EGFR. Pharmacologic inhibition of EGFR is sufficient to rewire the immune microenvironment. These results offer a readily accessible immunotherapy-sensitizing strategy for PDA.This article is highlighted in the In This Issue feature, p. 521.

RevDate: 2022-01-18

Li D, Li P, Peng M, et al (2022)

Transmission barrier of the blaKPC plasmid mediated by type I restriction-modification systems in Escherichia coli.

The Journal of antimicrobial chemotherapy pii:6510941 [Epub ahead of print].

BACKGROUND: Transportation of carbapenem-resistant plasmids contributes to carbapenem resistance in Gram-negative bacteria. KPC enzymes are the most clinically important enzymes among carbapenem-resistant Klebsiella pneumoniae, whereas the rate of blaKPC in Escherichia coli is low. The CRISPR-Cas system and restriction-modification system (R-M system) in bacteria defend against invading genomes. Currently, the role of the immune systems in the low rate of KPC-producing E. coli remains unclear.

OBJECTIVES: We investigated the relationship between immune systems and the low detection rate of blaKPC in E. coli.

METHODS: We searched for blaKPC among 1039 E. coli whole genomes available in GenBank using nucleotide BLAST. CRISPR-Cas systems and the R-M system were detected in all strains having the ST as blaKPC-positive strains. Nucleotide BLAST was used to search for protospacers on blaKPC plasmids. A conjugation assay was performed to determine whether the R-M system influences the acquisition of blaKPC plasmids by E. coli.

RESULTS: ST131 was the dominant ST of KPC-producing E. coli and IncN was the main plasmid type (12/32). CRISPR-Cas systems were frequently present in E. coli carrying blaKPC. Furthermore, CRISPR-Cas systems in E. coli didn't target plasmids with blaKPC. Type I R-M systems were rare in KPC-producing E. coli, but significantly over-represented in KPC-negative strains. E. coli DH5α with hsdR deletion accepted blaKPC-carrying plasmids, whereas those with hsdR complementation impeded blaKPC-carrying plasmid conjugation.

CONCLUSIONS: Horizontal transmission of blaKPC occurs among E. coli. The type I R-M system is associated with the defence against blaKPC plasmid transport into E. coli.

RevDate: 2022-01-18

Hasanzadeh A, Noori H, Jahandideh A, et al (2022)

Smart Strategies for Precise Delivery of CRISPR/Cas9 in Genome Editing.

ACS applied bio materials [Epub ahead of print].

The emergence of CRISPR/Cas technology has enabled scientists to precisely edit genomic DNA sequences. This approach can be used to modulate gene expression for the treatment of genetic disorders and incurable diseases such as cancer. This potent genome-editing tool is based on a single guide RNA (sgRNA) strand that recognizes the targeted DNA, plus a Cas nuclease protein for binding and processing the target. CRISPR/Cas has great potential for editing many genes in different types of cells and organisms both in vitro and in vivo. Despite these remarkable advances, the risk of off-target effects has hindered the translation of CRISPR/Cas technology into clinical applications. To overcome this hurdle, researchers have devised gene regulatory systems that can be controlled in a spatiotemporal manner, by designing special sgRNA, Cas, and CRISPR/Cas delivery vehicles that are responsive to different stimuli, such as temperature, light, magnetic fields, ultrasound (US), pH, redox, and enzymatic activity. These systems can even respond to dual or multiple stimuli simultaneously, thereby providing superior spatial and temporal control over CRISPR/Cas gene editing. Herein, we summarize the latest advances on smart sgRNA, Cas, and CRISPR/Cas nanocarriers, categorized according to their stimulus type (physical, chemical, or biological).

RevDate: 2022-01-17

Ramnarine SDBJ, Jayaraman J, A Ramsubhag (2022)

Comparative genomics of the black rot pathogen Xanthomonas campestris pv. campestris and non-pathogenic co-inhabitant Xanthomonas melonis from Trinidad reveal unique pathogenicity determinants and secretion system profiles.

PeerJ, 9:e12632 pii:12632.

Black-rot disease caused by the phytopathogen Xanthomonas campestris pv. campestris (Xcc) continues to have considerable impacts on the productivity of cruciferous crops in Trinidad and Tobago and the wider Caribbean region. While the widespread occurrence of resistance of Xcc against bactericidal agrochemicals can contribute to the high disease burdens, the role of virulence and pathogenicity features of local strains on disease prevalence and severity has not been investigated yet. In the present study, a comparative genomic analysis was performed on 6 pathogenic Xcc and 4 co-isolated non-pathogenic Xanthomonas melonis (Xmel) strains from diseased crucifer plants grown in fields with heavy chemical use in Trinidad. Native isolates were grouped into two known and four newly assigned ribosomal sequence types (rST). Mobile genetic elements were identified which belonged to the IS3, IS5 family, Tn3 transposon, resolvases, and tra T4SS gene clusters. Additionally, exogenous plasmid derived sequences with origins from other bacterial species were characterised. Although several instances of genomic rearrangements were observed, native Xcc and Xmel isolates shared a significant level of structural homology with reference genomes, Xcc ATCC 33913 and Xmel CFBP4644, respectively. Complete T1SS hlyDB, T2SS, T4SS vir and T5SS xadA, yapH and estA gene clusters were identified in both species. Only Xmel strains contained a complete T6SS but no T3SS. Both species contained a complex repertoire of extracellular cell wall degrading enzymes. Native Xcc strains contained 37 T3SS and effector genes but a variable and unique profile of 8 avr, 4 xop and 1 hpa genes. Interestingly, Xmel strains contained several T3SS effectors with low similarity to references including avrXccA1 (~89%), hrpG (~73%), hrpX (~90%) and xopAZ (~87%). Furthermore, only Xmel genomes contained a CRISPR-Cas I-F array, but no lipopolysaccharide wxc gene cluster. Xmel strains were confirmed to be non-pathogenic by pathogenicity assays. The results of this study will be useful to guide future research into virulence mechanisms, agrochemical resistance, pathogenomics and the potential role of the co-isolated non-pathogenic Xanthomonas strains on Xcc infections.

RevDate: 2022-01-16

Vyas P, Harish (2022)

Anti-CRISPR proteins as a therapeutic agent against drug-resistant bacteria.

Microbiological research, 257:126963 pii:S0944-5013(22)00003-9 [Epub ahead of print].

The continuous deployment of various antibiotics to treat multiple serious bacterial infections leads to multidrug resistance among the bacterial population. It has failed the standard treatment strategies through different antibacterial agents and serves as a significant threat to public health worldwide at devastating levels. The discovery of anti-CRISPR proteins catches the interest of researchers around the world as a promising therapeutic agent against drug-resistant bacteria. Anti-CRISPR proteins are known to inhibit bacterial CRISPR-Cas defense systems in multiple possible ways. The CRISPR-Cas nucleoprotein assembly provides adaptive immunity in bacteria against diverse categories of phage infections. Parallelly, phages also try to break the CRISPR-Cas barrier by producing anti-CRISPR proteins, leading to growth inhibition and bacterial lysis. This review begins with a brief description of the bacterial CRISPR-Cas system, followed by a detailed portrayal of anti-CRISPR proteins, including their discovery and evolution, mechanism of action, regulation of expression, and potential applications in the healthcare sector as an alternative therapeutic strategy to combat severe bacterial infections.

RevDate: 2022-01-17
CmpDate: 2022-01-17

Li PY, Li SQ, Gao SG, et al (2022)

A one-step platform for screening high-efficient and minimal off-target CRISPR/Cas13 crRNAs to eradicate SARS-CoV-2 virus for treatment of COVID-19 patients.

Medical hypotheses, 159:110754.

Coronavirus disease 2019 (COVID-19) is a new respiratory illness caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and now spreads globally. Currently, therapeutics and effective treatment options remain scarce and there is no proven drug to treat COVID-19. Targeting the positive-sense RNA genome and viral mRNAs of SARS-CoV-2 to simultaneously degrade viral genome templates for replication and viral mRNAs for essential gene expression would be a strategy to completely realize virus elimination. Type VI CRISPR enzymes Cas13 have recently been identified as programmable RNA-guided, RNA-targeting Cas proteins with nuclease activity that allows for RNA cleavage and degradation. The precise viral RNA detection and antiviral application of the CRISPR/Cas13 system depend on high-efficient and minimal off-target crRNAs. Although a computer-based algorithm has been applied for the design of crRNAs targeting SRAS-CoV-2, the experimental screening system to identify optimal crRNA is not available. We develop a one-step experimental screening system to identify high-efficient crRNAs with minimal off-target effects for CRISPR/Cas13-based SARS-CoV-2 elimination. This platform provides the foundation for CRISPR/Cas13-based diagnostics and therapeutics for COVID-19. This platform is versatile and could also be applied for crRNAs screening for other RNA viruses.

RevDate: 2022-01-18
CmpDate: 2022-01-18

Han C, Li W, Li Q, et al (2022)

CRISPR/Cas12a-Derived electrochemical aptasensor for ultrasensitive detection of COVID-19 nucleocapsid protein.

Biosensors & bioelectronics, 200:113922.

Fast, affordable, portable, and sensitive technology to detect COVID-19 is critical to address the current outbreak. Here, we present a CRISPR/Cas12a-derived electrochemical aptasensor for cost-effective, fast, and ultrasensitive COVID-19 nucleocapsid protein (Np) detection. First, an electrochemical sensing interface was fabricated by immobilizing methylene blue labeled poly adenines DNA sequence (polyA-MB electrochemical reporter) on a gold electrode surface. Second, an arched probe was prepared via hybridization of Np aptamer and an activator strand. In the presence of COVID-19 Np, the activator strand could be released from the arched probe due to the specific interaction between the target and the aptamer, which then activated the trans-cleavage activity of the CRISPR/Cas12a system. Subsequently, the polyA-MB reporters were cleaved from the electrode surface, decreasing the current of differential pulse voltammetry (DPV) at a potential of -0.27 V(vs. Ag/AgCl). The CRISPR/Cas12a-derived electrochemical aptasensor shows a highly efficient performance for COVID-19 Np detection in 50 pg mL-1 to 100 ng mL-1 with a limit of detection (LOD) low to 16.5 pg mL-1. Notably, the whole process of one test can be completed within 30 min. Simultaneously, the aptasensor displays a high selectivity to other proteins. The further measurements demonstrate that the aptasensor is robust in a natural system for point-of-care testing, such as in tap water, milk, or serum. The aptasensor is universal and expandable and holds great potential in the COVID-19 early diagnosis, environmental surveillance, food security, and other aspects.

RevDate: 2022-01-18
CmpDate: 2022-01-18

Chen A, Liao P, Li Q, et al (2021)

phytanoyl-CoA dioxygenase domain-containing protein 1 plays an important role in egg shell formation of silkworm (Bombyx mori).

PloS one, 16(12):e0261918.

Yun7Ge is a giant egg mutant found in the silkworm variety Yun7. In comparison with the giant mutant Ge, the eggs of Yun7Ge are larger. The number of laid eggs and hatching rate of Yun7Ge are reduced, which is not conducive to reproduction. In this work, the target gene controlling giant egg trait is located on the Z chromosome and was determined through genetic analysis. Transcriptome results showed that phytanoyl-CoA dioxygenase domain-containing protein 1 (PHYHD1) on the Z chromosome was silenced, and the 25 chorion genes on chromosome 2 were remarkably downregulated. Sequence analysis showed that the 73.5 kb sequence including the PHYHD1 was replaced by a ~3.0 kb sequence. After knocking out the PHYHD1 by using CRISPR/Cas9, the chorion genes were significantly downregulated. Hence, the silencing of PHYHD1 leads to the downregulation of many chorion protein genes, thus directly causing giant eggs.

RevDate: 2022-01-17
CmpDate: 2022-01-17

Moon HY, Sim GH, Kim HJ, et al (2022)

Assessment of Cre-lox and CRISPR-Cas9 as tools for recycling of multiple-integrated selection markers in Saccharomyces cerevisiae.

Journal of microbiology (Seoul, Korea), 60(1):18-30.

We evaluated the Cre-lox and CRISPR-Cas9 systems as marker-recycling tools in Saccharomyces cerevisiae recombinants containing multiple-integrated expression cassettes. As an initial trial, we constructed rDNA-nontranscribed spacer- or Ty4-based multiple integration vectors containing the URA3 marker flanked by the loxP sequence. Integrants harboring multiple copies of tHMG1 and NNV-CP expression cassettes were obtained and subsequently transformed with the Cre plasmid. However, the simultaneous pop-out of the expression cassettes along with the URA3 marker hampered the use of Cre-lox as a marker-recycling tool in multiple integrants. As an alternative, we constructed a set of CRISPR-Cas9-gRNA vectors containing gRNA targeted to auxotrophic marker genes. Transformation of multiple integrants of tHMG1 and NNV-CP cassettes by the Cas9-gRNA vector in the presence of the URA3 (stop) donor DNA fragments generated the Ura- transformants retaining multiple copies of the expression cassettes. CRISPR-Cas9-based inactivation led to the recycling of the other markers, HIS3, LEU2, and TRP1, without loss of expression cassettes in the recombinants containing multiple copies of tHMG1, NNV-CP, and SfBGL1 cassettes, respectively. Reuse of the same selection marker in marker-inactivated S. cerevisiae was validated by multiple integrations of the TrEGL2 cassette into the S. cerevisiae strain expressing SfBGL1. These results demonstrate that introducing stop codons into selection marker genes using the CRISPR-Cas9 system with donor DNA fragments is an efficient strategy for markerrecycling in multiple integrants. In particular, the continual reuse of auxotrophic markers would facilitate the construction of a yeast cell factory containing multiple copies of expression cassettes without antibiotic resistance genes.

RevDate: 2022-01-18
CmpDate: 2022-01-18

Son H, Park J, Hwang I, et al (2021)

Mg2+-dependent conformational rearrangements of CRISPR-Cas12a R-loop complex are mandatory for complete double-stranded DNA cleavage.

Proceedings of the National Academy of Sciences of the United States of America, 118(49):.

CRISPR-Cas12a, an RNA-guided DNA targeting endonuclease, has been widely used for genome editing and nucleic acid detection. As part of the essential processes for both of these applications, the two strands of double-stranded DNA are sequentially cleaved by a single catalytic site of Cas12a, but the mechanistic details that govern the generation of complete breaks in double-stranded DNA remain to be elucidated. Here, using single-molecule fluorescence resonance energy transfer assay, we identified two conformational intermediates that form consecutively following the initial cleavage of the nontarget strand. Specifically, these two intermediates are the result of further unwinding of the target DNA in the protospacer-adjacent motif (PAM)-distal region and the subsequent binding of the target strand to the catalytic site. Notably, the PAM-distal DNA unwound conformation was stabilized by Mg2+ ions, thereby significantly promoting the binding and cleavage of the target strand. These findings enabled us to propose a Mg2+-dependent kinetic model for the mechanism whereby Cas12a achieves cleavage of the target DNA, highlighting the presence of conformational rearrangements for the complete cleavage of the double-stranded DNA target.

RevDate: 2022-01-18
CmpDate: 2022-01-18

Oberhofer G, Ivy T, BA Hay (2021)

Gene drive that results in addiction to a temperature-sensitive version of an essential gene triggers population collapse in Drosophila.

Proceedings of the National Academy of Sciences of the United States of America, 118(49):.

One strategy for population suppression seeks to use gene drive to spread genes that confer conditional lethality or sterility, providing a way of combining population modification with suppression. Stimuli of potential interest could be introduced by humans, such as an otherwise benign virus or chemical, or occur naturally on a seasonal basis, such as a change in temperature. Cleave and Rescue (ClvR) selfish genetic elements use Cas9 and guide RNAs (gRNAs) to disrupt endogenous versions of an essential gene while also including a Rescue version of the essential gene resistant to disruption. ClvR spreads by creating loss-of-function alleles of the essential gene that select against those lacking it, resulting in populations in which the Rescue provides the only source of essential gene function. As a consequence, if function of the Rescue, a kind of Trojan horse now omnipresent in a population, is condition dependent, so too will be the survival of that population. To test this idea, we created a ClvR in Drosophila in which Rescue activity of an essential gene, dribble, requires splicing of a temperature-sensitive intein (TS-ClvRdbe). This element spreads to transgene fixation at 23 °C, but when populations now dependent on Ts-ClvRdb e are shifted to 29 °C, death and sterility result in a rapid population crash. These results show that conditional population elimination can be achieved. A similar logic, in which Rescue activity is conditional, could also be used in homing-based drive and to bring about suppression and/or killing of specific individuals in response to other stimuli.

RevDate: 2022-01-17
CmpDate: 2022-01-17

Hiraga H, Ishita Y, Chihara T, et al (2021)

Efficient visual screening of CRISPR/Cas9 genome editing in the nematode Pristionchus pacificus.

Development, growth & differentiation, 63(9):488-500.

CRISPR/Cas9 genome editing has been applied to a wide variety of organisms, including nematodes such as Caenorhabditis elegans and Pristionchus pacificus. In these nematodes, genome editing is achieved by microinjection of Cas9 protein and guide RNA into the hermaphrodite gonads. However, P. pacificus is less efficient in CRISPR/Cas9 genome editing and exogenous gene expression. Therefore, it takes considerable time and effort to screen for target mutants if there are no visual markers that indicate successful injection. To overcome this problem, co-injection markers (gRNA for Ppa-prl-1, which induces the roller phenotype, and Ppa-egl-20p::turboRFP, a plasmid expressing a fluorescent protein) have been developed in P. pacificus. By selecting worms with the roller phenotype or turboRFP expression, screening efficiency is substantially increased to obtain worms with desired mutations. Here, we describe a step-by-step protocol for the visual screening system for CRISPR/Cas9 genome editing in P. pacificus. We also describe technical tips for microinjection, which is difficult for beginners. This protocol will facilitate genome editing in P. pacificus and may be applied to other nematode species.

RevDate: 2022-01-17
CmpDate: 2022-01-17

Namba M, Kobayashi T, Koyano T, et al (2021)

GONAD: A new method for germline genome editing in mice and rats.

Development, growth & differentiation, 63(8):439-447.

Recent advances in the CRISPR/Cas9 system have demonstrated it to be an efficient gene-editing technology for various organisms. Laboratory mice and rats are widely used as common models of human diseases; however, the current standard method to create genome-engineered animals is laborious and involves three major steps: isolation of zygotes from females, ex vivo micromanipulation of zygotes, and implantation into pseudopregnant females. To circumvent this, we recently developed a novel method named Genome-editing via Oviductal Nucleic Acids Delivery (GONAD). This method does not require the ex vivo handling of embryos; instead, it can execute gene editing with just one step, via the delivery of a genome-editing mixture into embryos in the oviduct, by electroporation. Here, we present a further improvement of GONAD that is easily applicable to both mice and rats. It is a rapid, low-cost, and ethical approach fulfilling the 3R principles of animal experimentation: Reduction, Replacement, and Refinement. This method has been reconstructed and renamed as "improved GONAD (i-GONAD)" for mice, and "rat improved GONAD (rGONAD)" for rats.

RevDate: 2022-01-17
CmpDate: 2022-01-17

Xu X, Liu C, Wang Y, et al (2021)

Nanotechnology-based delivery of CRISPR/Cas9 for cancer treatment.

Advanced drug delivery reviews, 176:113891.

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 9) is a potent technology for gene-editing. Owing to its high specificity and efficiency, CRISPR/Cas9 is extensity used for human diseases treatment, especially for cancer, which involves multiple genetic alterations. Different concepts of cancer treatment by CRISPR/Cas9 are established. However, significant challenges remain for its clinical applications. The greatest challenge for CRISPR/Cas9 therapy is how to safely and efficiently deliver it to target sites in vivo. Nanotechnology has greatly contributed to cancer drug delivery. Here, we present the action mechanisms of CRISPR/Cas9, its application in cancer therapy and especially focus on the nanotechnology-based delivery of CRISPR/Cas9 for cancer gene editing and immunotherapy to pave the way for its clinical translation. We detail the difficult barriers for CRISIR/Cas9 delivery in vivo and discuss the relative solutions for encapsulation, target delivery, controlled release, cellular internalization, and endosomal escape.

RevDate: 2022-01-18
CmpDate: 2022-01-18

Zhao X, Henderson HJ, Wang T, et al (2021)

Deletion of Clusterin Protects Cochlear Hair Cells against Hair Cell Aging and Ototoxicity.

Neural plasticity, 2021:9979157.

Hearing loss is a debilitating disease that affects 10% of adults worldwide. Most sensorineural hearing loss is caused by the loss of mechanosensitive hair cells in the cochlea, often due to aging, noise, and ototoxic drugs. The identification of genes that can be targeted to slow aging and reduce the vulnerability of hair cells to insults is critical for the prevention of sensorineural hearing loss. Our previous cell-specific transcriptome analysis of adult cochlear hair cells and supporting cells showed that Clu, encoding a secreted chaperone that is involved in several basic biological events, such as cell death, tumor progression, and neurodegenerative disorders, is expressed in hair cells and supporting cells. We generated Clu-null mice (C57BL/6) to investigate its role in the organ of Corti, the sensory epithelium responsible for hearing in the mammalian cochlea. We showed that the deletion of Clu did not affect the development of hair cells and supporting cells; hair cells and supporting cells appeared normal at 1 month of age. Auditory function tests showed that Clu-null mice had hearing thresholds comparable to those of wild-type littermates before 3 months of age. Interestingly, Clu-null mice displayed less hair cell and hearing loss compared to their wildtype littermates after 3 months. Furthermore, the deletion of Clu is protected against aminoglycoside-induced hair cell loss in both in vivo and in vitro models. Our findings suggested that the inhibition of Clu expression could represent a potential therapeutic strategy for the alleviation of age-related and ototoxic drug-induced hearing loss.

RevDate: 2022-01-17
CmpDate: 2022-01-17

Zhang J, Khazalwa EM, Abkallo HM, et al (2021)

The advancements, challenges, and future implications of the CRISPR/Cas9 system in swine research.

Journal of genetics and genomics = Yi chuan xue bao, 48(5):347-360.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing technology has dramatically influenced swine research by enabling the production of high-quality disease-resistant pig breeds, thus improving yields. In addition, CRISPR/Cas9 has been used extensively in pigs as one of the tools in biomedical research. In this review, we present the advancements of the CRISPR/Cas9 system in swine research, such as animal breeding, vaccine development, xenotransplantation, and disease modeling. We also highlight the current challenges and some potential applications of the CRISPR/Cas9 technologies.

RevDate: 2022-01-17
CmpDate: 2022-01-17

Grandhi TSP, To J, Romero A, et al (2021)

High-throughput CRISPR-mediated 3D enrichment platform for functional interrogation of chemotherapeutic resistance.

Biotechnology and bioengineering, 118(8):3187-3199.

Cancer is a disease of somatic mutations. These cellular mutations compete to dominate their microenvironment and dictate the disease outcome. While a therapeutic approach to target-specific oncogenic driver mutations helps to manage the disease, subsequent molecular evolution of tumor cells threatens to overtake therapeutic progress. There is a need for rapid, high-throughput, unbiased in vitro discovery screening platforms that capture the native complexities of the tumor and rapidly identify mutations that confer chemotherapeutic drug resistance. Taking the example of the CDK4/6 inhibitor (CDK4/6i) class of drugs, we show that the pooled in vitro CRISPR screening platform enables rapid discovery of drug resistance mutations in a three-dimensional (3D) setting. Gene-edited cancer cell clones assembled into an organotypic multicellular tumor spheroid (MCTS), exposed to CDK4/6i caused selection and enrichment of the most drug-resistant phenotypes, detectable by next-gen sequencing after a span of 28 days. The platform was sufficiently sensitive to enrich for even a single drug-resistant cell within a large, drug-responsive complex 3D tumor spheroid. The genome-wide 3D CRISPR-mediated knockout screen (>18,000 genes) identified several genes whose disruptions conferred resistance to CDK4/6i. Furthermore, multiple novel candidate genes were identified as top hits only in the microphysiological 3D enrichment assay platform and not the conventional 2D assays. Taken together, these findings suggest that including phenotypic 3D resistance profiling in decision trees could improve discovery and reconfirmation of drug resistance mechanisms and afford a platform for exploring noncell autonomous interactions, selection pressures, and clonal competition.

RevDate: 2022-01-17
CmpDate: 2022-01-17

Qiao X, Gao Y, Li J, et al (2021)

Sensitive analysis of single nucleotide variation by Cas13d orthologs, EsCas13d and RspCas13d.

Biotechnology and bioengineering, 118(8):3037-3045.

RNA-guided CRISPR (RNA-targeting clustered regularly interspaced short palindromic repeats) effector Cas13d is the smallest Class II subtype VI proteins identified so far. Here, two recently identified Cas13d effectors from Eubacterium siraeum (Es) and Ruminococcus sp. (Rsp) were characterized and applied for sensitive nucleic acid detection. We demonstrated that the special target triggered collateral cleavage of these two Cas13d orthologs could provide rapid target RNA detection in picomolar range and then the tolerance for mismatch between crRNA and target RNA was characterized as well. Finally, an additional single mismatch was introduced into crRNA to enhance the two Cas13d orthologs mediated detection of low variant allele fraction, 0.1% T790M. Overall, this study demonstrated that both EsCas13d and RspCas13d could robustly detect target RNA carrying special single-nucleotide variation with high specificity and sensitivity, thereby providing newly qualified machinery in toolbox for efficient molecular diagnostics.

RevDate: 2022-01-18
CmpDate: 2022-01-18

Zhu L, Yang X, Li J, et al (2021)

Leptin gene-targeted editing in ob/ob mouse adipose tissue based on the CRISPR/Cas9 system.

Journal of genetics and genomics = Yi chuan xue bao, 48(2):134-146.

Gene therapy has become the most effective treatment for monogenic diseases. Congenital LEPTIN deficiency is a rare autosomal recessive monogenic obesity syndrome caused by mutations in the Leptin gene. Ob/ob mouse is a monogenic obesity model, which carries a homozygous point mutation of C to T in Exon 2 of the Leptin gene. Here, we attempted to edit the mutated Leptin gene in ob/ob mice preadipocytes and inguinal adipose tissues using CRISPR/Cas9 to correct the C to T mutation and restore the production of LEPTIN protein by adipocytes. The edited preadipocytes exhibit a correction of 5.5% of Leptin alleles and produce normal LEPTIN protein when differentiated into mature adipocytes. The ob/ob mice display correction of 1.67% of Leptin alleles, which is sufficient to restore the production and physiological functions of LEPTIN protein, such as suppressing appetite and alleviating insulin resistance. Our study suggests CRISPR/Cas9-mediated in situ genome editing as a feasible therapeutic strategy for human monogenic diseases, and paves the way for further research on efficient delivery system in potential future clinical application.

RevDate: 2022-01-18
CmpDate: 2022-01-18

Chen M, Shi H, Gou S, et al (2021)

In vivo genome editing in mouse restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers.

Genome medicine, 13(1):57.

BACKGROUND: Mutations in the DMD gene encoding dystrophin-a critical structural element in muscle cells-cause Duchenne muscular dystrophy (DMD), which is the most common fatal genetic disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing is a promising strategy for permanently curing DMD.

METHODS: In this study, we developed a novel strategy for reframing DMD mutations via CRISPR-mediated large-scale excision of exons 46-54. We compared this approach with other DMD rescue strategies by using DMD patient-derived primary muscle-derived stem cells (DMD-MDSCs). Furthermore, a patient-derived xenograft (PDX) DMD mouse model was established by transplanting DMD-MDSCs into immunodeficient mice. CRISPR gene editing components were intramuscularly delivered into the mouse model by adeno-associated virus vectors.

RESULTS: Results demonstrated that the large-scale excision of mutant DMD exons showed high efficiency in restoring dystrophin protein expression. We also confirmed that CRISPR from Prevotella and Francisella 1(Cas12a)-mediated genome editing could correct DMD mutation with the same efficiency as CRISPR-associated protein 9 (Cas9). In addition, more than 10% human DMD muscle fibers expressed dystrophin in the PDX DMD mouse model after treated by the large-scale excision strategies. The restored dystrophin in vivo was functional as demonstrated by the expression of the dystrophin glycoprotein complex member β-dystroglycan.

CONCLUSIONS: We demonstrated that the clinically relevant CRISPR/Cas9 could restore dystrophin in human muscle cells in vivo in the PDX DMD mouse model. This study demonstrated an approach for the application of gene therapy to other genetic diseases.

RevDate: 2022-01-17
CmpDate: 2022-01-17

Chan W, Gottschalk RA, Yao Y, et al (2021)

Efficient Immune Cell Genome Engineering with Enhanced CRISPR Editing Tools.

ImmunoHorizons, 5(2):117-132 pii:5/2/117.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based methods have revolutionized genome engineering and the study of gene-phenotype relationships. However, modifying cells of the innate immune system, especially macrophages, has been challenging because of cell pathology and low targeting efficiency resulting from nucleic acid activation of intracellular sensors. Likewise, lymphocytes of the adaptive immune system are difficult to modify using CRISPR-enhanced homology-directed repair because of inefficient or toxic delivery of donor templates using transient transfection methods. To overcome these challenges and limitations, we modified existing tools and developed three alternative methods for CRISPR-based genome editing using a hit-and-run transient expression strategy, together with a convenient system for promoting gene expression using CRISPRa. Overall, our CRISPR tools and strategies designed to tackle both murine and human immune cell genome engineering provide efficient alternatives to existing methods and have wide application not only in terms of hematopoietic cells but also other mammalian cell types of interest.

RevDate: 2022-01-18
CmpDate: 2022-01-18

Niklasson CU, Fredlund E, Monni E, et al (2021)

Hypoxia inducible factor-2α importance for migration, proliferation, and self-renewal of trunk neural crest cells.

Developmental dynamics : an official publication of the American Association of Anatomists, 250(2):191-236.

BACKGROUND: The neural crest is a transient embryonic stem cell population. Hypoxia inducible factor (HIF)-2α is associated with neural crest stem cell appearance and aggressiveness in tumors. However, little is known about its role in normal neural crest development.

RESULTS: Here, we show that HIF-2α is expressed in trunk neural crest cells of human, murine, and avian embryos. Knockdown as well as overexpression of HIF-2α in vivo causes developmental delays, induces proliferation, and self-renewal capacity of neural crest cells while decreasing the proportion of neural crest cells that migrate ventrally to sympathoadrenal sites. Reflecting the in vivo phenotype, transcriptome changes after loss of HIF-2α reveal enrichment of genes associated with cancer, invasion, epithelial-to-mesenchymal transition, and growth arrest.

CONCLUSIONS: Taken together, these results suggest that expression levels of HIF-2α must be strictly controlled during normal trunk neural crest development and that dysregulated levels affects several important features connected to stemness, migration, and development.

RevDate: 2022-01-17
CmpDate: 2022-01-17

Kampmann M (2020)

CRISPR-based functional genomics for neurological disease.

Nature reviews. Neurology, 16(9):465-480.

Neurodegenerative, neurodevelopmental and neuropsychiatric disorders are among the greatest public health challenges, as many lack disease-modifying treatments. A major reason for the absence of effective therapies is our limited understanding of the causative molecular and cellular mechanisms. Genome-wide association studies are providing a growing catalogue of disease-associated genetic variants, and the next challenge is to elucidate how these variants cause disease and to translate this understanding into therapies. This Review describes how new CRISPR-based functional genomics approaches can uncover disease mechanisms and therapeutic targets in neurological diseases. The bacterial CRISPR system can be used in experimental disease models to edit genomes and to control gene expression levels through CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa). These genetic perturbations can be implemented in massively parallel genetic screens to evaluate the functional consequences for human cells. CRISPR screens are particularly powerful in combination with induced pluripotent stem cell technology, which enables the derivation of differentiated cell types, such as neurons and glia, and brain organoids from cells obtained from patients. Modelling of disease-associated changes in gene expression via CRISPRi and CRISPRa can pinpoint causal changes. In addition, genetic modifier screens can be used to elucidate disease mechanisms and causal determinants of cell type-selective vulnerability and to identify therapeutic targets.

RevDate: 2022-01-15

Yu Y, Li W, Gu X, et al (2021)

Inhibition of CRISPR-Cas12a trans-cleavage by lead (II)-induced G-quadruplex and its analytical application.

Food chemistry, 378:131802 pii:S0308-8146(21)02808-9 [Epub ahead of print].

In this work, the inhibition of clustered regularly interspaced short palindromic repeats (CRISPR) - CRISPR associated protein (Cas) trans-cleavage by Pb2+-induced G-quadruplex has been firstly explored to detect Pb2+ in tea beverage and milk. In absence of Pb2+, the Na+-induced G-quadruplex can be cleaved by CRISPR-Cas12a. In contrast, Pb2+ can competitively combine with G-quadruplex, resulting in its conformational changes and resistance to trans-cleavage of CRISPR-Cas12a. Therefore, the fluorescence resonance energy transfer can happen. Pb2+ can be detected in a linear range from 100 nM to 5 µM with a lowest detection limit of 2.6 nM and a relative standard deviation of 4.32%. In summary, this work not only provides a new method for Pb2+ detection based on its induced G-quadruplex inhibition on CRISPR-Cas12a trans-cleavage, but also broadens the application of CRISPR-Cas system for heavy metal analysis in the field of food safety.

RevDate: 2022-01-15

Vera LNP, Schuh RS, Fachel FNS, et al (2022)

Brain and visceral gene editing of mucopolysaccharidosis I mice by nasal delivery of the CRISPR/Cas9 system.

The journal of gene medicine [Epub ahead of print].

INTRODUCTION: Mucopolysaccharidosis type I (MPS I) is an inherited disease caused by deficiency of the enzyme alpha-L-iduronidase (IDUA). MPS I affects several tissues, including the brain, leading to cognitive impairment in the severe form of the disease. Currently available treatments do not reach the brain. Therefore, in this study, we performed nasal administration (NA) of liposomal complexes carrying two plasmids encoding for the CRISPR/Cas9 system and for the IDUA gene targeting the ROSA26 locus, aiming at brain delivery in MPS I mice.

METHODS: Liposomes were prepared by microfluidization and the plasmids were complexed to the formulations by adsorption. Physicochemical characterization of the formulations and complexes, in vitro permeation, and mucoadhesion in porcine nasal mucosa (PNM) were assessed. We performed NA repeatedly for 30 days in young MPS I mice, which were euthanized at 6 months of age after performing behavioral tasks, and biochemical and molecular aspects were evaluated.

RESULTS: Monodisperse mucoadhesive complexes around 110nm, which are able to efficiently permeate the PNM. In animals the treatment led to a modest increase in IDUA activity in the lung, heart and brain areas, with reduction of glycosaminoglycan (GAG) levels in serum, urine, tissues and brain cortex. Furthermore, treated mice showed improvement in behavioral tests, suggesting prevention of the cognitive damage.

CONCLUSION: Non-viral gene editing performed through nasal route represents a potential therapeutic alternative for the somatic and neurologic symptoms of MPS I and possibly to other neurological disorders.

RevDate: 2022-01-15

Sun ML, Shi TQ, Lin L, et al (2022)

Advancing Yarrowia lipolytica as a superior biomanufacturing platform by tuning gene expression using promoter engineering.

Bioresource technology pii:S0960-8524(22)00046-3 [Epub ahead of print].

Yarrowia lipolytica is recognized as an excellent non-conventional yeast in the field of biomanufacturing, where it is used as a host to produce oleochemicals, terpenes, organic acids, polyols and recombinant proteins. Consequently, metabolic engineering of this yeast is becoming increasingly popular to advance it as a superior biomanufacturing platform, of which promoters are the most basic elements for tuning gene expression. Endogenous promoters of Yarrowia lipolytica were reviewed, which are the basis for promoter engineering. The engineering strategies, such as hybrid promoter engineering, intron enhancement promoter engineering, and transcription factor-based inducible promoter engineering are described. Additionally, the applications of Yarrowia lipolytica promoter engineering to rationally reconstruct biosynthetic gene clusters and improve the genome-editing efficiency of the CRISPR-Cas systems were reviewed. Finally, research needs and future directions for promoter engineering are also discussed in this review.

RevDate: 2022-01-15

Abdolhosseini M, Zandsalimi F, Moghaddam FS, et al (2022)

A review on colorimetric assays for DNA virus detection.

Journal of virological methods pii:S0166-0934(22)00008-8 [Epub ahead of print].

Early detection is one of the ways to deal with DNA virus widespread prevalence, and it is necessary to know new diagnostic methods and techniques. Colorimetric assays are one of the most advantageous methods in detecting viruses. These methods are based on color change, which can be seen either with the naked eye or with special devices. The aim of this study is to introduce and evaluate effective colorimetric methods based on amplification, nanoparticle, CRISPR/Cas, and Lateral flow in the diagnosis of DNA viruses and to discuss the effectiveness of each of the updated methods. Compared to the other methods, colorimetric assays are preferred for faster detection, high efficiency, cheaper cost, and high sensitivity and specificity. It is expected that the spread of these viruses can be prevented by identifying and developing new methods.

RevDate: 2022-01-14
CmpDate: 2022-01-14

Bumpus TW, Huang S, Tei R, et al (2021)

Click chemistry-enabled CRISPR screening reveals GSK3 as a regulator of PLD signaling.

Proceedings of the National Academy of Sciences of the United States of America, 118(48):.

Enzymes that produce second messengers are highly regulated. Revealing the mechanisms underlying such regulation is critical to understanding both how cells achieve specific signaling outcomes and return to homeostasis following a particular stimulus. Pooled genome-wide CRISPR screens are powerful unbiased approaches to elucidate regulatory networks, their principal limitation being the choice of phenotype selection. Here, we merge advances in bioorthogonal fluorescent labeling and CRISPR screening technologies to discover regulators of phospholipase D (PLD) signaling, which generates the potent lipid second messenger phosphatidic acid. Our results reveal glycogen synthase kinase 3 as a positive regulator of protein kinase C and PLD signaling. More generally, this work demonstrates how bioorthogonal, activity-based fluorescent tagging can expand the power of CRISPR screening to uncover mechanisms regulating specific enzyme-driven signaling pathways in mammalian cells.

RevDate: 2022-01-14
CmpDate: 2022-01-14

Zhang JY, Niu TC, Lin GM, et al (2022)

A CRISPR-Based Method for Constructing Conditional Mutations of Essential Genes in Cyanobacteria.

Methods in molecular biology (Clifton, N.J.), 2377:143-157.

Cyanobacteria, a group of diverse bacteria capable of oxygenic photosynthesis, are excellent models for investigating many important cellular processes, such as photosynthesis, nitrogen fixation, and prokaryotic cell differentiation. They also have great potential to become the next-generation cell factories for sustainable biosynthesis of valuable products. However, genetic manipulation in cyanobacteria is not as convenient as in other model bacteria. Particularly, handling essential genes in cyanobacteria has been difficult due to the lack of appropriate tools, limiting our understanding of many important cellular functions encoded by them. We recently develop a CRISPR-based method for constructing the conditional mutants of cyanobacterial essential genes by engineering the ribosome binding site to a theophylline-responsive riboswitch. Here, we provide the details of this method. The principle of this method could be used to construct conditional mutants in a wide range of bacterial species.

RevDate: 2022-01-14
CmpDate: 2022-01-14

Liao X, Xing XH, C Zhang (2022)

New Method for Genome-Scale Functional Genomic Study in Bacteria with Superior Performance: CRISPR Interference Screen.

Methods in molecular biology (Clifton, N.J.), 2377:123-141.

High-throughput genetic screens based on CRISPR/Cas9 technology are powerful tools to genome-wide identify gene function and genotype-phenotype association. Here, we describe a detailed protocol for conducting and evaluating pooled CRISPR screens interfering with gene expression in Escherichia coli. We provide step-by-step instructions for guide RNA library design and construction, genome-scale screening and next-generation sequencing data processing. This tool outperforms transposon sequencing (Tn-seq) with similar library sizes and short gene length. The workflow can be used in follow-up studies implemented in other bacteria systems.

RevDate: 2022-01-14
CmpDate: 2022-01-14

Nishimura K, T Fukagawa (2022)

A Simple Method that Combines CRISPR and AID to Quickly Generate Conditional Knockouts for Essential Genes in Various Vertebrate Cell Lines.

Methods in molecular biology (Clifton, N.J.), 2377:109-122.

Cells with a loss-of-function mutation in a gene (knockout cells) are powerful tools for characterizing the function of such gene product. However, for essential genes, conditional knockout cell lines must be generated. The auxin-inducible degron (AID) technique enables us to conditionally and rapidly deplete a target protein from various eukaryotic cell lines. A combination of CRISPR-/Cas9-based gene editing and AID technique allows us to generate AID-based conditional knockout cell lines. Using these two techniques, we recently proposed a simple and quick way to generate conditional knockout cells for essential genes. In this chapter, we introduce the reader to the experimental procedures to generate these AID-based conditional knockout cell lines.

RevDate: 2022-01-14
CmpDate: 2022-01-14

DeHart L, Yockey OP, J Bakke (2022)

Identification of Essential Genes Using Sequential CRISPR and siRNA Screens.

Methods in molecular biology (Clifton, N.J.), 2377:89-107.

Genome-wide CRISPR and siRNA screening methodologies are powerful tools that are aptly suited to the discovery of essential genes. In this chapter, we outline our methods to conduct sequential CRISPR and siRNA screens to quickly and efficiently identify essential genes within a collection of cell lines. The utilization of both screening methodologies provides a pipeline that minimizes costs and time while enabling the robust detection of candidate genes.

RevDate: 2022-01-14
CmpDate: 2022-01-14

Wensing L, RS Shapiro (2022)

Design and Generation of a CRISPR Interference System for Genetic Repression and Essential Gene Analysis in the Fungal Pathogen Candida albicans.

Methods in molecular biology (Clifton, N.J.), 2377:69-88.

Studying life-threatening fungal pathogens such as Candida albicans is of critical importance, yet progress can be hindered by challenges associated with manipulating these pathogens genetically. CRISPR-based technologies have significantly improved our ability to manipulate the genomes of countless organisms, including fungal pathogens such as C. albicans. CRISPR interference (CRISPRi) is a modified variation of CRISPR technology that enables the targeted genetic repression of specific genes of interest and can be used as a technique for studying essential genes. We recently developed tools to enable CRISPRi in C. albicans and the repression of essential genes in this fungus. Here, we describe a protocol for CRISPRi in C. albicans, including the design of the single-guide RNAs (sgRNAs) for targeting essential genes, the high-efficiency cloning of sgRNAs into C. albicans-optimized CRISPRi plasmids, transformation into fungal strains, and testing to monitor the repression capabilities of these constructs. Together, this protocol will illuminate efficient strategies for targeted genetic repression of essential genes in C. albicans using a novel CRISPRi platform.

RevDate: 2022-01-14
CmpDate: 2022-01-14

Huggler KS, Rossiter NJ, Flickinger KM, et al (2022)

CRISPR/Cas9 Screening to Identify Conditionally Essential Genes in Human Cell Lines.

Methods in molecular biology (Clifton, N.J.), 2377:29-42.

Forward genetic screens across hundreds of cancer cell lines have started to define the genetic dependencies of proliferating human cells. However, most such screens have been performed in vitro with little consideration into how medium composition might affect gene essentiality. This protocol describes a method to use CRISPR/Cas9-based loss-of-function screens to ask how gene essentiality in human cell lines varies with medium composition. First, a single-guide RNA (sgRNA) library is packaged into lentivirus, and an optimal infection titer is determined for the target cells. Following selection, genomic DNA (gDNA) is extracted from an aliquot of the transduced cells. The remaining transduced cells are then screened in at least two distinct cell culture media. At the conclusion of the screening period, gDNA is collected from each cell population. Next, high-throughput sequencing is used to determine sgRNA barcode abundances from the initial and each of the final populations. Finally, an analytical pipeline is used to identify medium-essential candidate genes from these screen results.

RevDate: 2022-01-14
CmpDate: 2022-01-14

Mair B, Aregger M, Tong AHY, et al (2022)

A Method to Map Gene Essentiality of Human Pluripotent Stem Cells by Genome-Scale CRISPR Screens with Inducible Cas9.

Methods in molecular biology (Clifton, N.J.), 2377:1-27.

Human pluripotent stem cells (hPSCs) have the capacity for self-renewal and differentiation into most cell types and, in contrast to widely used cell lines, are karyotypically normal and non-transformed. Hence, hPSCs are considered the gold-standard system for modelling diseases, especially in the field of regenerative medicine. Despite widespread research use of hPSCs and induced pluripotent stem cells (iPSCs), the systematic understanding of pluripotency and lineage differentiation mechanisms are still incomplete. Before tackling the complexities of lineage differentiation with genetic screens, it is critical to catalogue the general genetic requirements for cell fitness and proliferation in the pluripotent state and assess their plasticity under commonly used culture conditions.We describe a method to map essential genetic determinants of hPSC fitness and pluripotency, herein defined as cell reproduction, by genome-scale loss-of-function CRISPR screens in an inducible S. pyogenes Cas9 H1 hPSC line. To address questions of context-dependent gene essentiality, we include protocols for screening hPSCs cultured on feeder cells and laminin, two commonly used growth substrates. This method establishes parameters for genome-wide screens in hPSCs, making human stem cells amenable for functional genomics approaches to facilitate investigation of hPSC biology.

RevDate: 2022-01-14
CmpDate: 2022-01-14

Krause WC, Rodriguez R, Gegenhuber B, et al (2021)

Oestrogen engages brain MC4R signalling to drive physical activity in female mice.

Nature, 599(7883):131-135.

Oestrogen depletion in rodents and humans leads to inactivity, fat accumulation and diabetes1,2, underscoring the conserved metabolic benefits of oestrogen that inevitably decrease with age. In rodents, the preovulatory surge in 17β-oestradiol (E2) temporarily increases energy expenditure to coordinate increased physical activity with peak sexual receptivity. Here we report that a subset of oestrogen-sensitive neurons in the ventrolateral ventromedial hypothalamic nucleus (VMHvl)3-7 projects to arousal centres in the hippocampus and hindbrain, and enables oestrogen to rebalance energy allocation in female mice. Surges in E2 increase melanocortin-4 receptor (MC4R) signalling in these VMHvl neurons by directly recruiting oestrogen receptor-α (ERα) to the Mc4r gene. Sedentary behaviour and obesity in oestrogen-depleted female mice were reversed after chemogenetic stimulation of VMHvl neurons expressing both MC4R and ERα. Similarly, a long-term increase in physical activity is observed after CRISPR-mediated activation of this node. These data extend the effect of MC4R signalling - the most common cause of monogenic human obesity8 - beyond the regulation of food intake and rationalize reported sex differences in melanocortin signalling, including greater disease severity of MC4R insufficiency in women9. This hormone-dependent node illuminates the power of oestrogen during the reproductive cycle in motivating behaviour and maintaining an active lifestyle in women.

RevDate: 2022-01-14
CmpDate: 2022-01-14

Zhang W, Xu L, Liu Q, et al (2021)

Enzymatic recombinase amplification coupled with CRISPR-Cas12a for ultrasensitive, rapid, and specific Porcine circovirus 3 detection.

Molecular and cellular probes, 59:101763.

Porcine circovirus type 3 (PCV3) is a disease associated with porcine dermatitis and nephrotic syndrome (PDNS) that has caused significant economic losses to swine herds since its discovery in 2016. To develop a simple, on-site, rapid, and sensitive assay to combat the spread of PCV3, we optimized the CRISPR/Cas12a (also known as Cpf1) system combined with enzymatic recombinase amplification (ERA) nucleic acid amplification to diagnose PCV3. The results showed that the ERA-CRISPR/Cas12a reaction could detect PCV3 within 1 h in genomic DNA harboring a minimum of seven copies. Additionally, we confirmed no cross-reactivity with PCV2, PCV4, or other porcine viruses, revealing the good specificity of this technique. These results demonstrated the ability of ERA-CRISPR/Cas12a to detect DNA at the single-molecule level and provide a rapid, simple, ultrasensitive, one-pot point-of-care test for PCV3 and suggest its potential for a variety of nucleic acid detection applications.

RevDate: 2022-01-14
CmpDate: 2022-01-14

Tilson SG, Morell CM, Lenaerts AS, et al (2021)

Modeling PNPLA3-Associated NAFLD Using Human-Induced Pluripotent Stem Cells.

Hepatology (Baltimore, Md.), 74(6):2998-3017.

BACKGROUND AND AIMS: NAFLD is a growing public health burden. However, the pathogenesis of NAFLD has not yet been fully elucidated, and the importance of genetic factors has only recently been appreciated. Genomic studies have revealed a strong association between NAFLD progression and the I148M variant in patatin-like phospholipase domain-containing protein 3 (PNPLA3). Nonetheless, very little is known about the mechanisms by which this gene and its variants can influence disease development. To investigate these mechanisms, we have developed an in vitro model that takes advantage of the unique properties of human-induced pluripotent stem cells (hiPSCs) and the CRISPR/CAS9 gene editing technology.

APPROACH AND RESULTS: We used isogenic hiPSC lines with either a knockout (PNPLA3KO) of the PNPLA3 gene or with the I148M variant (PNPLA3I148M) to model PNPLA3-associated NAFLD. The resulting hiPSCs were differentiated into hepatocytes, treated with either unsaturated or saturated free fatty acids to induce NAFLD-like phenotypes, and characterized by various functional, transcriptomic, and lipidomic assays. PNPLA3KO hepatocytes showed higher lipid accumulation as well as an altered pattern of response to lipid-induced stress. Interestingly, loss of PNPLA3 also caused a reduction in xenobiotic metabolism and predisposed PNPLA3KO cells to be more susceptible to ethanol-induced and methotrexate-induced toxicity. The PNPLA3I148M cells exhibited an intermediate phenotype between the wild-type and PNPLA3KO cells.

CONCLUSIONS: Together, these results indicate that the I148M variant induces a loss of function predisposing to steatosis and increased susceptibility to hepatotoxins.

RevDate: 2022-01-14
CmpDate: 2022-01-14

Lau MSH, Sheng L, Zhang Y, et al (2021)

Development of a Suite of Tools for Genome Editing in Parageobacillus thermoglucosidasius and Their Use to Identify the Potential of a Native Plasmid in the Generation of Stable Engineered Strains.

ACS synthetic biology, 10(7):1739-1749.

The relentless rise in the levels of atmospheric greenhouse gases caused by the exploitation of fossil fuel necessitates the development of more environmentally friendly routes to the manufacture of chemicals and fuels. The exploitation of a fermentative process that uses a thermophilic chassis represents an attractive option. Its use, however, is hindered by a dearth of genetic tools. Here we expand on those available for the engineering of the industrial chassis Parageobacillus thermoglucosidasius through the assembly and testing of a range of promoters, ribosome binding sites, reporter genes, and the implementation of CRISPR/Cas9 genome editing based on two different thermostable Cas9 nucleases. The latter were used to demonstrate that the deletion of the two native plasmids carried by P. thermoglucosidasius, pNCI001 and pNCI002, either singly or in combination, had no discernible effects on the overall phenotypic characteristics of the organism. Through the CRISPR/Cas9-mediated insertion of the gene encoding a novel fluorescent reporter, eCGP123, we showed that pNCI001 exhibited a high degree of segregational stability. As the relatively higher copy number of pNCI001 led to higher levels of eCGP123 expression than when the same gene was integrated into the chromosome, we propose that pNCI001 represents the preferred option for the integration of metabolic operons when stable commercial strains are required.

RevDate: 2022-01-14
CmpDate: 2022-01-14

Smith CW, Kachwala MJ, Nandu N, et al (2021)

Recognition of DNA Target Formulations by CRISPR-Cas12a Using a dsDNA Reporter.

ACS synthetic biology, 10(7):1785-1791.

CRISPR-Cas12a is a powerful platform for DNA-based diagnostics. The detection scheme relies on unselective shredding of a fluorescent ssDNA reporter upon target DNA recognition. To extend the reporter library beyond ssDNAs, we discovered a fluorescent reporter type using a dsDNA template. In this design, the fluorescence of the dsDNA reporter is quenched via contact-quenching mechanism. Upon detection, the quenched fluorescence recovers with the activation Cas12a complex. Here, we compared the probing performance of two dsDNA reporters with two ssDNA reporters. The rate of the Cas12a trans-cleavage reaction was studied using one of the dsDNA reporters under different settings. The detection of different sizes of dsDNA or ssDNA targets was studied systematically under three different temperatures. Lower thresholds for ssDNA and dsDNA target size were identified. The mismatch tolerance and target specificity were examined for both ssDNA and dsDNA targets, separately. The probing performance of the dsDNA reporter was evaluated in a random DNA pool with and without target strands. We report that dsDNA can serve as a tunable fluorescence reporter template expanding the toolbox for Cas12a-based diagnostics.

RevDate: 2022-01-14
CmpDate: 2022-01-14

Brault J, Liu T, Bello E, et al (2021)

CRISPR-targeted MAGT1 insertion restores XMEN patient hematopoietic stem cells and lymphocytes.

Blood, 138(26):2768-2780.

XMEN disease, defined as "X-linked MAGT1 deficiency with increased susceptibility to Epstein-Barr virus infection and N-linked glycosylation defect," is a recently described primary immunodeficiency marked by defective T cells and natural killer (NK) cells. Unfortunately, a potentially curative hematopoietic stem cell transplantation is associated with high mortality rates. We sought to develop an ex vivo targeted gene therapy approach for patients with XMEN using a CRISPR/Cas9 adeno-associated vector (AAV) to insert a therapeutic MAGT1 gene at the constitutive locus under the regulation of the endogenous promoter. Clinical translation of CRISPR/Cas9 AAV-targeted gene editing (GE) is hampered by low engraftable gene-edited hematopoietic stem and progenitor cells (HSPCs). Here, we optimized GE conditions by transient enhancement of homology-directed repair while suppressing AAV-associated DNA damage response to achieve highly efficient (>60%) genetic correction in engrafting XMEN HSPCs in transplanted mice. Restored MAGT1 glycosylation function in human NK and CD8+ T cells restored NK group 2 member D (NKG2D) expression and function in XMEN lymphocytes for potential treatment of infections, and it corrected HSPCs for long-term gene therapy, thus offering 2 efficient therapeutic options for XMEN poised for clinical translation.

RevDate: 2022-01-14
CmpDate: 2022-01-14

Balboa D, Iworima DG, TJ Kieffer (2021)

Human Pluripotent Stem Cells to Model Islet Defects in Diabetes.

Frontiers in endocrinology, 12:642152.

Diabetes mellitus is characterized by elevated levels of blood glucose and is ultimately caused by insufficient insulin production from pancreatic beta cells. Different research models have been utilized to unravel the molecular mechanisms leading to the onset of diabetes. The generation of pancreatic endocrine cells from human pluripotent stem cells constitutes an approach to study genetic defects leading to impaired beta cell development and function. Here, we review the recent progress in generating and characterizing functional stem cell-derived beta cells. We summarize the diabetes disease modeling possibilities that stem cells offer and the challenges that lie ahead to further improve these models.

RevDate: 2022-01-14
CmpDate: 2022-01-14

Umemura K, Ohtsuki S, Nagaoka M, et al (2021)

Critical contribution of macrophage scavenger receptor 1 to the uptake of nanostructured DNA by immune cells.

Nanomedicine : nanotechnology, biology, and medicine, 34:102386.

Despite the efficient uptake of polypod-like nanostructured DNA, or polypodna, by macrophage-like RAW264.7 and other immune cells, the detailed mechanism has not been fully elucidated. Our previous study using HEK-Blue hTLR9 cells showed that transfection of macrophage scavenger receptor 1 (MSR1) increased the uptake of tetrapod-like structured DNA. Here, we investigated the involvement of MSR1 in the structure-dependent uptake of polypodna. Transfection of MSR1 to HEK-Blue hTLR9 cells pod number-dependently increased the uptake of polypodna, and its knockout in RAW264.7 cells reduced the uptake and subsequent cytokine release. To examine the binding of DNA with MSR1, biotinylated DNA added to RAW264.7 cells was cross-linked with cell surface proteins. Then, MSR1 cross-linked with polypodna, but not with single-stranded DNA. Similar results were obtained with murine primary immune cells. Taken together, MSR1 discriminates between simple and nanostructured DNAs and plays a dominant role in the efficient uptake of polypodna by immune cells.

RevDate: 2022-01-14
CmpDate: 2022-01-14

Bandara AB, Drake JC, James CC, et al (2021)

Complex I protein NDUFS2 is vital for growth, ROS generation, membrane integrity, apoptosis, and mitochondrial energetics.

Mitochondrion, 58:160-168.

Complex I is the largest and most intricate of the protein complexes of mitochondrial electron transport chain (ETC). This L-shaped enzyme consists of a peripheral hydrophilic matrix domain and a membrane-bound orthogonal hydrophobic domain. The interfacial region between these two arms is known to be critical for binding of ubiquinone moieties and has also been shown to be the binding site of Complex I inhibitors. Knowledge on specific roles of the ETC interfacial region proteins is scarce due to lack of knockout cell lines and animal models. Here we mutated nuclear encoded NADH dehydrogenase [ubiquinone] iron-sulfur protein 2 (NDUFS2), one of three protein subunits of the interfacial region, in a human embryonic kidney cell line 293 using a CRISPR/Cas9 procedure. Disruption of NDUFS2 significantly decreased cell growth in medium, Complex I specific respiration, glycolytic capacity, ATP pool and cell-membrane integrity, but significantly increased Complex II respiration, ROS generation, apoptosis, and necrosis. Treatment with idebenone, a clinical benzoquinone currently being investigated in other indications, partially restored growth, ATP pool, and oxygen consumption of the mutant. Overall, our results suggest that NDUFS2 is vital for growth and metabolism of mammalian cells, and respiratory defects of NDUFS2 dysfunction can be partially corrected with treatment of an established mitochondrial therapeutic candidate. This is the first report to use CRISPR/Cas9 approach to construct a knockout NDUFS2 cell line and use the constructed mutant to evaluate the efficacy of a known mitochondrial therapeutic to enhance bioenergetic capacity.

RevDate: 2022-01-14
CmpDate: 2022-01-14

Salick MR, Lubeck E, Riesselman A, et al (2021)

The future of cerebral organoids in drug discovery.

Seminars in cell & developmental biology, 111:67-73.

Until the discovery of human embryonic stem cells and human induced pluripotent stem cells, biotechnology companies were severely limited in the number of human tissues that they could model in large-scale in vitro studies. Until this point, companies have been limited to immortalized cancer lines or a small number of primary cell types that could be extracted and expanded. Nowadays, protocols continue to be developed in the stem cell field, enabling researchers to model an ever-growing library of cell types in controlled, large-scale screens. One differentiation method in particular- cerebral organoids- shows substantial potential in the field of neuroscience and developmental neurobiology. Cerebral organoid technology is still in an early phase of development, and there are several challenges that are currently being addressed by academic and industrial researchers alike. Here we briefly describe some of the early adopters of cerebral organoids, several of the challenges that they are likely facing, and various technologies that are currently being implemented to overcome them.

RevDate: 2022-01-13

Taha EA, Lee J, A Hotta (2022)

Delivery of CRISPR-Cas tools for in vivo genome editing therapy: Trends and challenges.

Journal of controlled release : official journal of the Controlled Release Society pii:S0168-3659(22)00027-X [Epub ahead of print].

The discovery of clustered regularly interspaced short palindromic repeats (CRISPR) genome editing technology opened the door to provide a versatile approach for treating multiple diseases. Promising results have been shown in numerous pre-clinical studies and clinical trials. However, a safe and effective method to deliver genome-editing components is still a key challenge for in vivo genome editing therapy. Adeno-associated virus (AAV) is one of the most commonly used vector systems to date, but immunogenicity against capsid, liver toxicity at high dose, and potential genotoxicity caused by off-target mutagenesis and genomic integration remain unsolved. Recently developed transient delivery systems, such as virus-like particle (VLP) and lipid nanoparticle (LNP), may solve some of the issues. This review summarizes existing in vivo delivery systems and possible solutions to overcome their limitations. Also, we highlight the ongoing clinical trials for in vivo genome editing therapy and recently developed genome editing tools for their potential applications.

RevDate: 2022-01-13

Barrangou R, LA Marraffini (2022)

Turning CRISPR on with antibiotics.

Cell host & microbe, 30(1):12-14.

CRISPR-Cas systems have the ability to integrate invasive DNA sequences to build adaptive immunity in bacteria. In this issue Dimitriu et al. show bacteriostatic antibiotics prompt CRISPR acquisition events, illustrating how environmental conditions affect complex dynamics between host and virus and the corresponding biological and genetic arms race.

RevDate: 2022-01-13

Lewis IC, Yan Y, GC Finnigan (2021)

Analysis of a Cas12a-based gene-drive system in budding yeast.

Access microbiology, 3(12):000301 pii:000301.

The discovery and adaptation of CRISPR/Cas systems within molecular biology has provided advances across biological research, agriculture and human health. Genomic manipulation through use of a CRISPR nuclease and programmed guide RNAs has become a common and widely accessible practice. The identification and introduction of new engineered variants and orthologues of Cas9 as well as alternative CRISPR systems such as the type V group have provided additional molecular options for editing. These include distinct PAM requirements, staggered DNA double-strand break formation, and the ability to multiplex guide RNAs from a single expression construct. Use of CRISPR/Cas has allowed for the construction and testing of a powerful genetic architecture known as a gene drive within eukaryotic model systems. Our previous work developed a drive within budding yeast using Streptococcus pyogenes Cas9. Here, we installed the type V Francisella novicida Cas12a (Cpf1) nuclease gene and its corresponding guide RNA to power a highly efficient artificial gene drive in diploid yeast. We examined the consequence of altering guide length or introduction of individual mutational substitutions to the crRNA sequence. Cas12a-dependent gene-drive function required a guide RNA of at least 18 bp and could not tolerate most changes within the 5' end of the crRNA.

RevDate: 2022-01-13

Chen S, Wan L, Wei DD, et al (2022)

High Prevalence and Fitness of IncFrepB Carrying qnrS1 in Hypervirulent Klebsiella pneumoniae Isolates.

Microbial drug resistance (Larchmont, N.Y.) [Epub ahead of print].

Objective: This study aimed to reveal the prevalence and fitness of qnrS1-carrying plasmids in hypervirulent Klebsiella pneumoniae (hvKP) isolates. Materials and Methods: Two hundred ninety-nine hvKP strains carrying qnrS1 were collected and screened for resistance genes using PCR and sequencing. The location of qnrS1 and rmpA2 was identified by Southern blotting. The transferability and fitness of qnrS1-carrying plasmids were analyzed by conjugation experiments and plasmid stability assay. Result: In 299 hvKP isolates, the most frequently detected capsular serotype was K64 (81.9%, 245/299), followed by K1 (4.7%, 14/299) and K2 (3.7%, 11/299). All K64-hvKP were sequence type (ST) 11. The qnrS1 and rmpA2 gene mainly was located on the ∼70-210 kb IncFrepB and ∼170-220 kb IncFIB plasmid, respectively. QnrS1-carrying plasmids could be transferred into Escherichia coli J53. However, the plasmid was transferred at a low rate of 13.4% (40/299). The 40 donor isolates belong to 4 STs-ST11, ST700, ST592, and ST86, and none contains the CRISPR-Cas loci. CRISPR-Cas loci were mainly found in ST23 K. pneumoniae. The relative fitness (RF) of qnrS1-carrying plasmids in ST86 and ST11 (cotransfer with blaTEM-1 genes) was more than one and enhanced during cultivation, especially in ST86. However, the RF of qnrS1-carrying plasmids in ST592 and ST700 showed a high fitness cost. Whole-genome sequencing showed that the qnrS1-carrying plasmids in ST86 harbored more maintenance modules (SOS inhibitor protein psiB, parA, and parB partition systems) and insertion sequence (IS) elements (IS91, IS481-like, IS1380), indicating that the qnrS1-carrying plasmid in ST86 is more stable than the other types of qnrS1-carrying plasmids. Conclusion: QnrS1-carrying IncFrepB plasmids were highly prevalent and show polymorphism in hvKP strains. The qnrS1-carrying IncFrepB plasmid in ST86 hvKP should be highlighted due to its remarkable adaptability advantages.

RevDate: 2022-01-13
CmpDate: 2022-01-13

Aoshima T, Kobayashi Y, Takagi H, et al (2021)

Modification of improved-genome editing via oviductal nucleic acids delivery (i-GONAD)-mediated knock-in in rats.

BMC biotechnology, 21(1):63.

BACKGROUND: Improved genome-editing via oviductal nucleic acids delivery (i-GONAD) is a new technology that facilitates in situ genome-editing of mammalian zygotes exiting the oviductal lumen. The i-GONAD technology has been developed for use in mice, rats, and hamsters; however, oligonucleotide (ODN)-based knock-in (KI) is more inefficient in rats than mice. To improve the efficiency of i-GONAD in rats we examined KI efficiency using three guide RNAs (gRNA), crRNA1, crRNA2 and crRNA3. These gRNAs recognize different portions of the target locus, but also overlap each other in the target locus. We also examined the effects of commercially available KI -enhancing drugs (including SCR7, L755,507, RS-1, and HDR enhancer) on i-GONAD-mediated KI efficiency.

RESULTS: The KI efficiency in rat fetuses generated after i-GONAD with crRNA2 and single-stranded ODN was significantly higher (24%) than crRNA1 (5%; p < 0.05) or crRNA3 (0%; p < 0.01). The KI efficiency of i-GONAD with triple gRNAs was 11%. These findings suggest that KI efficiency largely depends on the type of gRNA used. Furthermore, the KI efficiency drugs, SCR7, L755,507 and HDR enhancer, all of which are known to enhance KI efficiency, increased KI efficiency using the i-GONAD with crRNA1 protocol. In contrast, only L755,507 (15 μM) increased KI efficiency using the i-GONAD with crRNA2 protocol. None of them were significantly different.

CONCLUSIONS: We attempted to improve the KI efficiency of i-GONAD in rats. We demonstrated that the choice of gRNA is important for determining KI efficiency and insertion and deletion rates. Some drugs (e.g. SCR7, L755,507 and HDR enhancer) that are known to increase KI efficiency in culture cells were found to be effective in i-GONAD in rats, but their effects were limited.

RevDate: 2022-01-13
CmpDate: 2022-01-13

Chatterjee N, X Zhang (2021)

CRISPR approach in environmental chemical screening focusing on population variability.

The Journal of toxicological sciences, 46(11):499-507.

A significant barrier to include population variability in risk assessment is our incomplete understanding of inter-individual variability and the differential susceptibility to environmental exposures induced adverse outcomes. By combining genome editing tools with the population diversity model, this article intended to highlight a potential strategy to identify and characterize the inter-individual variability factors, the determinant gene anchoring to a particular phenotype. The goal could be achieved by integrating the perturbed CRISPR-based unbiased functional genomics screening, genome-wide or a focused subset of genes, in a population-based in vitro model system (such as the lymphoblastoid cell lines, LCL, available from HapMap and 1000 Genomes project). Then data can be translated to genetic variability and individual (or subpopulation) susceptibility by incorporating ethnicity and corresponding genome-wide association studies (GWAS) with functional genomics screening results. This approach can provide complementary data for next-generation risk assessment, in particular, for environmental stressors. The current paper outlined the previous work conducted with a population-based in vitro model system, perturbed CRISPR-based functional toxicogenomic screening of environmental chemicals, and finally, the potential strategies to combine these two platforms with their opportunities and challenges to achieve a mechanistic understanding of population variability.

RevDate: 2022-01-13
CmpDate: 2022-01-13

Dubey A, Kumar A, Malla MA, et al (2021)

Approaches for the amelioration of adverse effects of drought stress on crop plants.

Frontiers in bioscience (Landmark edition), 26(10):928-947.

Climate change, water scarcity, population growth, and food shortage are some of the threatening challenges being faced in today's world. Among different types of stresses, drought stress presents a persistent challenge for global food production, however, its harshness and intensity are supposed to expand in the imminent future. The most striking effects of drought stress on plants are stunted growth, severe damage to photosynthetic apparatus, reduction in photosynthesis, reduction in seed germination, and nutrient uptake. To deal with the destructive effect of drought stress on plants, it is necessary to consider its effects, mechanisms of action, the agronomic and genetic basis for sustainable management. Therefore, there is an urgent need for sustainable solutions to cope up with the negative impact of drought stress. This review focuses on the detrimental effects of drought stress on plants' morphological, physiological, and biochemical characteristics and recommends suitable drought management techniques to reduce the severity of drought stress. We summarize the effect of drought stress on physiological and biochemical parameters (such as germination, photosynthesis, biomass, water status, and nutrient uptake) and yield. Overall, in this article, we have reviewed the role of different phytohormones, osmolytes, exogenous compounds, proteins, plant growth-promoting microbes (PGPM), omics approaches, and genome editing technologies like clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR-Cas9) in alleviating drought effects in plants. We also proposed that developing drought-tolerant plant varieties requires the combined use of biotechnological and agronomic approaches and cutting-edge genome editing (GE) tools.

RevDate: 2022-01-13
CmpDate: 2022-01-13

Huang S, Yan Y, Su F, et al (2021)

Research progress in gene editing technology.

Frontiers in bioscience (Landmark edition), 26(10):916-927.

As a tool for modifying the genome, gene editing technology has developed rapidly in recent years, especially in the past two years. With the emergence of new gene editing technologies, such as transposon editing tools, numerous advancements have been made including precise editing of the genome, double base editing, and pilot editing. This report focuses on the development of gene editing tools in recent years, elaborates the progress made in classic editing tools, base editor and other new editing tools, and provides insights into challenges and opportunities.

RevDate: 2022-01-13
CmpDate: 2022-01-13

Zou YL, Ye AJ, Liu S, et al (2021)

Expansion of targetable sites for the ribonucleoprotein-based CRISPR/Cas9 system in the silkworm Bombyx mori.

BMC biotechnology, 21(1):54.

BACKGROUND: With the emergence of CRISPR/Cas9 technology, multiple gene editing procedures became available for the silkworm. Although binary transgene-based methods have been widely used to generate mutants, delivery of the CRISPR/Cas9 system via DNA-free ribonucleoproteins offers several advantages. However, the T7 promoter that is widely used in the ribonucleoprotein-based method for production of sgRNAs in vitro requires a 5' GG motif for efficient initiation. The resulting transcripts bear a 5' GG motif, which significantly constrains the number of targetable sites in the silkworm genome.

RESULTS: In this study, we used the T7 promoter to add two supernumerary G residues to the 5' end of conventional (perfectly matched) 20-nucleotide sgRNA targeting sequences. We then asked if sgRNAs with this structure can generate mutations even if the genomic target does not contain corresponding GG residues. As expected, 5' GG mismatches depress the mutagenic activity of sgRNAs, and a single 5' G mismatch has a relatively minor effect. However, tests involving six sgRNAs targeting two genes show that the mismatches do not eliminate mutagenesis in vivo, and the efficiencies remain at useable levels. One sgRNA with a 5' GG mismatch at its target performed mutagenesis more efficiently than a conventional sgRNA with 5' matched GG residues at a second target within the same gene. Mutations generated by sgRNAs with 5' GG mismatches are also heritable. We successfully obtained null mutants with detectable phenotypes from sib-mated mosaics after one generation.

CONCLUSIONS: In summary, our method improves the utility and flexibility of the ribonucleoprotein-based CRISPR/Cas9 system in silkworm.

RevDate: 2022-01-13
CmpDate: 2022-01-13

Takeda I, Araki M, Ishiguro KI, et al (2021)

Gene trapping reveals a new transcriptionally active genome element: The chromosome-specific clustered trap region.

Genes to cells : devoted to molecular & cellular mechanisms, 26(11):874-890.

Nearly half of the human genome consists of repetitive sequences such as long interspersed nuclear elements. The relationship between these repeating sequences and diseases has remained unclear. Gene trapping is a useful technique for disrupting a gene and expressing a reporter gene by using the promoter activity of the gene. The analysis of trapped genes revealed a new genome element-the chromosome-specific clustered trap (CSCT) region. For any examined sequence within this region, an equivalent was found using the BLAT of the University of California, Santa Cruz (UCSC) Genome Browser. CSCT13 mapped to chromosome 13 and contained only three genes. To elucidate its in vivo function, the whole CSCT13 region (1.6 Mbp) was deleted using the CRISPR/Cas9 system in mouse embryonic stem cells, and subsequently, a CSCT13 knockout mouse line was established. The rate of homozygotes was significantly lower than expected according to Mendel's laws. In addition, the number of offspring obtained by mating homozygotes was significantly smaller than that obtained by crossing controls. Furthermore, CSCT13 might have an effect on meiotic homologous recombination. This study identifies a transcriptionally active CSCT with an important role in mouse development.

RevDate: 2022-01-13
CmpDate: 2022-01-13

Liu N, Zhou L, Qu J, et al (2021)

Recent Advances in CRISPR/Cas9 Directed Base Editing.

Current gene therapy, 21(4):327-337.

Recently, CRISPR-based techniques have significantly improved our ability to make desired changes and regulations in various genomes. Among them, targeted base editing is one of the most powerful techniques in making precise genomic editing. Base editing enabled the irreversible conversion of a specific single DNA base, from C to T or and from A to G, in desired genomic loci. This technique has important implications in the study of human genetic diseases, considering that many of them resulted from point mutations. More importantly, the high efficiency of these editing tools also provided great promise in clinical applications. In this review, we discuss the recent progress and challenges of base editing tools.

RevDate: 2022-01-12

Rodrigues C, Desai S, Passet V, et al (2022)

Genomic evolution of the globally disseminated multidrug-resistant Klebsiella pneumoniae clonal group 147.

Microbial genomics, 8(1):.

The rapid emergence of multidrug-resistant Klebsiella pneumoniae is being driven largely by the spread of specific clonal groups (CGs). Of these, CG147 includes 7-gene multilocus sequence typing (MLST) sequence types (STs) ST147, ST273 and ST392. CG147 has caused nosocomial outbreaks across the world, but its global population dynamics remain unknown. Here, we report a pandrug-resistant ST147 clinical isolate from India (strain DJ) and define the evolution and global emergence of CG147. Antimicrobial-susceptibility testing following European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines and genome sequencing (Illumina and Oxford Nanopore Technologies, Unicycler assembly) were performed on strain DJ. Additionally, we collated 217 publicly available CG147 genomes [National Center for Biotechnology Information (NCBI), May 2019]. CG147 evolution was inferred within a temporal phylogenetic framework (beast) based on a recombination-free sequence alignment (Roary/Gubbins). Comparative genomic analyses focused on resistance and virulence genes and other genetic elements (BIGSdb, Kleborate, PlasmidFinder, phaster, ICEfinder and CRISPRCasFinder). Strain DJ had a pandrug-resistance phenotype. Its genome comprised the chromosome, seven plasmids and one linear phage-plasmid. Four carbapenemase genes were detected: bla NDM-5 and two copies of bla OXA-181 in the chromosome, and a second copy of bla NDM-5 on an 84 kb IncFII plasmid. CG147 genomes carried a mean of 13 acquired resistance genes or mutations; 63 % carried a carbapenemase gene and 83 % harboured bla CTX-M. All CG147 genomes presented GyrA and ParC mutations and a common subtype I-E CRISPR-Cas system. ST392 and ST273 emerged in 2005 and 1995, respectively. ST147, the most represented phylogenetic branch, was itself divided into two main clades with distinct capsular loci: KL64 (74 %, DJ included, emerged in 1994 and disseminated worldwide, with carbapenemases varying among world regions) and KL10 (20 %, emerged in 2002, predominantly found in Asian countries, associated with carbapenemases NDM and OXA-48-like). Furthermore, subclades within ST147-KL64 differed at the yersiniabactin locus, OmpK35/K36 mutations, plasmid replicons and prophages. The absence of IncF plasmids in some subclades was associated with a possible activity of a CRISPR-Cas system. K. pneumoniae CG147 comprises pandrug-resistant or extensively resistant isolates, and carries multiple and diverse resistance genes and mobile genetic elements, including chromosomal bla NDM-5. Its emergence is being driven by the spread of several phylogenetic clades marked by their own genomic features and specific temporo-spatial dynamics. These findings highlight the need for precision surveillance strategies to limit the spread of particularly concerning CG147 subsets.

RevDate: 2022-01-12

Deb S, Choudhury A, Kharbyngar B, et al (2022)

Applications of CRISPR/Cas9 technology for modification of the plant genome.

Genetica [Epub ahead of print].

The CRISPR/Cas (Clustered regularly interspaced short palindromic repeats/ CRISPR associated protein 9) system was discovered in bacteria and archea as an acquired immune response to protect the cells from infection. This technology has now evolved to become an efficient genome editing tool, and is replacing older gene editing technologies. This technique uses programmable sgRNAs to guide the Cas9 endonuclease to the target DNA location. sgRNA is a vital component of the CRISPR technology, since without it the Cas nuclease cannot reach to its target location. Over the years, many tools have been developed for designing sgRNAs, the details of which have been extensively reviewed here. It has proven to be a promising tool in the field of genetic engineering and has successfully generated many plant varieties with better and desirable qualities. In the present review, we attempted to collect,collate and summarize information related to the development of CRISPR/Cas9 system as a tool and subsequently into a technique having a wide array of applications in the field of plant genome editing in attaining desirable traits like resistance to various diseases, nutritional enhancement etc. In addition, the probable future prospects and the various bio-safety concerns associated with CRISPR gene editing technology have been discussed in detail.

RevDate: 2022-01-12

Hao Y, Wang Q, Li J, et al (2022)

Double nicking by RNA-directed Cascade-nCas3 for high-efficiency large-scale genome engineering.

Open biology, 12(1):210241.

New CRISPR-based genome editing technologies are developed to continually drive advances in life sciences, which, however, are predominantly derived from systems of Type II CRISPR-Cas9 and Type V CRISPR-Cas12a for eukaryotes. Here we report a novel CRISPR-n(nickase)Cas3 genome editing tool established upon a Type I-F system. We demonstrate that nCas3 variants can be created by alanine-substituting any catalytic residue of the Cas3 helicase domain. While nCas3 overproduction via plasmid shows severe cytotoxicity, an in situ nCas3 introduces targeted double-strand breaks, facilitating genome editing without visible cell killing. By harnessing this CRISPR-nCas3 in situ gene insertion, nucleotide substitution and deletion of genes or genomic DNA stretches can be consistently accomplished with near-100% efficiencies, including simultaneous removal of two large genomic fragments. Our work describes the first establishment of a CRISPR-nCas3-based genome editing technology, thereby offering a simple, yet useful approach to convert the naturally most abundantly occurring Type I systems into advanced genome editing tools to facilitate high-throughput prokaryotic engineering.

RevDate: 2022-01-12
CmpDate: 2022-01-12

Seronick E, Son J, Michael C, et al (2022)

CRISPR/Cas9 genome editing system confirms centriolin's role in cytokinesis.

BMC research notes, 15(1):8.

OBJECTIVE: In addition to its function as the microtubule organizing center of the cell, the centrosome has functions in many other cellular processes including primary cilia formation, DNA damage checkpoints, and cell cycle progression. But the role of individual components of the centrosome in these processes remains unclear. Previous studies used siRNA (small interfering RNA) to "knock down" protein levels of the centrosome component centriolin, resulting in failed cytokinesis. Since this approach was transient, only targeting centriolin at the mRNA level, we sought to confirm these findings by permanently disrupting the gene encoding centriolin using the CRISPR/Cas9 system of genome editing.

RESULTS: This study provides evidence that the CRISPR/Cas9 system is capable of effectively reducing centriolin protein levels in the cell. Furthermore, this disruption leads to a failure of cytokinesis that is reminiscent of the phenotype previously reported for the siRNA-mediated disruption of centriolin. Furthermore, no additional defects in cell division were observed, consistent with results seen with previous siRNA studies. We conclude that the CRISPR/Cas9 system is an effective means of permanently removing the cellular pools of centriolin and that the disruption of centriolin at both the mRNA level and genomic level lead to similar cell division defects.

RevDate: 2022-01-12
CmpDate: 2022-01-12

Kruasuwan W, Puseenam A, Tanapongpipat S, et al (2021)

Multiplexed CRISPR-mediated engineering of protein secretory pathway genes in the thermotolerant methylotrophic yeast Ogataea thermomethanolica.

PloS one, 16(12):e0261754.

CRISPR multiplex gRNA systems have been employed in genome engineering in various industrially relevant yeast species. The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 is an alternative host for heterologous protein production. However, the limited secretory capability of this yeast is a bottleneck for protein production. Here, we refined CRISPR-based genome engineering tools for simultaneous mutagenesis and activation of multiple protein secretory pathway genes to improve heterologous protein secretion. We demonstrated that multiplexed CRISPR-Cas9 mutation of up to four genes (SOD1, VPS1, YPT7 and YPT35) in one single cell is practicable. We also developed a multiplexed CRISPR-dCas9 system which allows simultaneous activation of multiple genes in this yeast. 27 multiplexed gRNA combinations were tested for activation of three genes (SOD1, VPS1 and YPT7), three of which were demonstrated to increase the secretion of fungal xylanase and phytase up to 29% and 41%, respectively. Altogether, our study provided a toolkit for mutagenesis and activation of multiple genes in O. thermomethanolica, which could be useful for future strain engineering to improve heterologous protein production in this yeast.

RevDate: 2022-01-12
CmpDate: 2022-01-12

Yokochi Y, Fukushi Y, Wakabayashi KI, et al (2021)

Oxidative regulation of chloroplast enzymes by thioredoxin and thioredoxin-like proteins in Arabidopsis thaliana.

Proceedings of the National Academy of Sciences of the United States of America, 118(51):.

Thioredoxin (Trx) is a protein that mediates the reducing power transfer from the photosynthetic electron transport system to target enzymes in chloroplasts and regulates their activities. Redox regulation governed by Trx is a system that is central to the adaptation of various chloroplast functions to the ever-changing light environment. However, the factors involved in the opposite reaction (i.e., the oxidation of various enzymes) have yet to be revealed. Recently, it has been suggested that Trx and Trx-like proteins could oxidize Trx-targeted proteins in vitro. To elucidate the in vivo function of these proteins as oxidation factors, we generated mutant plant lines deficient in Trx or Trx-like proteins and studied how the proteins are involved in oxidative regulation in chloroplasts. We found that f-type Trx and two types of Trx-like proteins, Trx-like 2 and atypical Cys His-rich Trx (ACHT), seemed to serve as oxidation factors for Trx-targeted proteins, such as fructose-1,6-bisphosphatase, Rubisco activase, and the γ-subunit of ATP synthase. In addition, ACHT was found to be involved in regulating nonphotochemical quenching, which is the mechanism underlying the thermal dissipation of excess light energy. Overall, these results indicate that Trx and Trx-like proteins regulate chloroplast functions in concert by controlling the redox state of various photosynthesis-related proteins in vivo.

RevDate: 2022-01-12
CmpDate: 2022-01-12

Qiu Z, Park A, Wang L, et al (2022)

The RGD (Arg-Gly-Asp) is a potential cell-binding motif of UNC-52/PERLECAN.

Biochemical and biophysical research communications, 586:143-149.

UNC-52/perlecan is a basement membrane (BM) proteoglycan playing an essential role in the muscle cell attachment of C. elegans. The UNC-52 protein contains two RGD (Arg-Gly-Asp) motifs in domains III and IV, a well-characterized tripeptide known for binding to mammalian β integrin. To investigate the role of the RGD motif in UNC-52/perlecan, we created two mutations in the 2021RGD2023 motif: one mutation changed the RGD to an RGE, and the other deleted the RGD motif. The RGE2023 caused defective actin filaments and aberrant localization of PAT-3 β integrin and TLN-1/talin. Additionally, the in-frame deletion of RGD2023 resulted in a paralyzed and arrested at two-fold embryonic stages (Pat) phenotype, which is the identical phenotype of the pat-3 β integrin null allele. These results indicate that RGD2023 is a potential ligand for cell binding and is essential for development and survival. Furthermore, our analysis reveals that the RGD of an invertebrate BM molecule is a potential cell-binding motif, suggesting that the function of the RGD motif is conserved among species.

RevDate: 2022-01-12
CmpDate: 2022-01-12

Racharaks R, Arnold W, J Peccia (2021)

Development of CRISPR-Cas9 knock-in tools for free fatty acid production using the fast-growing cyanobacterial strain Synechococcus elongatus UTEX 2973.

Journal of microbiological methods, 189:106315.

Synechococcus elongatus UTEX 2973 has one of the fastest measured doubling time of cyanobacteria making it an important candidate for metabolic engineering. Traditional genetic engineering methods, which rely on homologous recombination, however, are inefficient, labor-intensive, and time-consuming due to the oligoploidy or polyploidy nature of cyanobacteria and the reliance on unique antibiotic resistance markers. CRISPR-Cas9 has emerged as an effective and versatile editing platform in a wide variety of organisms, but its application for cyanobacterial engineering is limited by the inherent toxicity of Cas9 resulting in poor transformation efficiencies. Here, we demonstrated that a single-plasmid CRISPR-Cas9 system, pCRISPOmyces-2, can effectively knock-in a truncated thioesterase gene from Escherichia coli to generate free fatty acid (FFA) producing mutants of Syn2973. To do so, three parameters were evaluated on the effect of generating recipient colonies after conjugation with pCRISPOmyces-2-based plasmids: 1) a modified conjugation protocol termed streaked conjugation, 2) the deletion of the gene encoding RecJ exonuclease, and 3) single guide RNA (sgRNA) sequence. With the use of the streaked conjugation protocol and a ΔrecJ mutant strain of Syn2973, the conjugation efficiency for the pCRISPomyces-2 plasmid could be improved by 750-fold over the wildtype (WT) for a conjugation efficiency of 2.0 × 10-6 transconjugants/recipient cell. While deletion of the RecJ exonuclease alone increased the conjugation efficiency by 150-fold over the WT, FFA generation was impaired in FFA-producing mutants with the ΔrecJ background, and the large number of poor FFA-producing isolates indicated the potential increase in spontaneous mutation rates. The sgRNA sequence was found to be critical in achieving the desired CRISPR-Cas9-mediated knock-in mutation as the sgRNA impacts conjugation efficiency, likelihood of homogenous recombinants, and free fatty acid production in engineered strains.

RevDate: 2022-01-12
CmpDate: 2022-01-12

Sun H, Hodgkinson CP, Pratt RE, et al (2021)

CRISPR/Cas9 Mediated Deletion of the Angiotensinogen Gene Reduces Hypertension: A Potential for Cure?.

Hypertension (Dallas, Tex. : 1979), 77(6):1990-2000.

[Figure: see text].

RevDate: 2022-01-12
CmpDate: 2022-01-12

Huang C, Li G, Wu J, et al (2021)

Identification of pathogenic variants in cancer genes using base editing screens with editing efficiency correction.

Genome biology, 22(1):80.

BACKGROUND: Millions of nucleotide variants are identified through cancer genome sequencing and it is clinically important to identify the pathogenic variants among them. By introducing base substitutions at guide RNA target regions in the genome, CRISPR-Cas9-based base editors provide the possibility for evaluating a large number of variants in their genomic context. However, the variability in editing efficiency and the complexity of outcome mapping are two existing problems for assigning guide RNA effects to variants in base editing screens.

RESULTS: To improve the identification of pathogenic variants, we develop a framework to combine base editing screens with sgRNA efficiency and outcome mapping. We apply the method to evaluate more than 9000 variants across all the exons of BRCA1 and BRCA2 genes. Our efficiency-corrected scoring model identifies 910 loss-of-function variants for BRCA1/2, including 151 variants in the noncoding part of the genes such as the 5' untranslated regions. Many of them are identified in cancer patients and are reported as "benign/likely benign" or "variants of uncertain significance" by clinicians. Our data suggest a need to re-evaluate their clinical significance, which may be helpful for risk assessment and treatment of breast and ovarian cancer.

CONCLUSIONS: Our results suggest that base editing screens with efficiency correction is a powerful strategy to identify pathogenic variants in a high-throughput manner. Applying this strategy to assess variants in both coding and noncoding regions of the genome could have a direct impact on the interpretation of cancer variants.

RevDate: 2022-01-11

Zhao L, Zhao T, Yang X, et al (2022)

IL-37 blocks gouty inflammation by shaping macrophages into a non-inflammatory phagocytic phenotype.

Rheumatology (Oxford, England) pii:6500277 [Epub ahead of print].

OBJECTIVE: Interleukin (IL)-37 is a natural suppressor of inflammation. Macrophages play an important role in acute gout flare by dominating the inflammation and spontaneous relief. We have reported IL-37 could limit runaway inflammation in gout. Here we focus on whether IL-37 inhibits gouty inflammation by altering macrophage functions and how it does.

METHODS: Macrophage functions were evaluated in terms of phagocytosis, pyroptosis, polarization, and metabolism. Phagocytosis and polarization of macrophages were detected by side scattering and double-labelling iNOS/Arg-1 using flow cytometry, respectively. Transcription of pyroptosis-related molecules was detected by qPCR. Metabolomics was performed by liquid chromatograph mass spectrometer. Human IL-37 knock-in mice and a model with point mutation (S9A) at mouse Gsk3b locus were created by CRISPR/Cas-mediated genome engineering. MSU was injected into paws and peritoneal cavity to model acute gout. Vernier caliper was used to measure the thickness of the paws. The mice paws and human synovium tissues or tophi were collected for pathological staining. Peritoneal fluid of mice was used to enrich macrophages to detect polarization.

RESULTS: IL-37 promoted non-inflammatory phagocytic activity of macrophages, by enhancing phagocytosis of MSU, reducing pyroptosis-related proteins transcription and inflammatory cytokines releasing, protecting mitochondrial function, and mediating metabolic reprogramming in MSU-treated THP-1 cells. These multifaceted roles of IL-37 were partly depended on the mediation of glycogen synthase kinase-3β (GSK-3β).

CONCLUSIONS: Our study revealed that IL-37 could shape macrophages into a "silent" non-inflammatory phagocytic fashion. IL-37 may become a potentially valuable treatment option for patients of chronic gout, especially for those with tophi.

RevDate: 2022-01-11

Kiryushkin AS, Ilina EL, Guseva ED, et al (2021)

Hairy CRISPR: Genome Editing in Plants Using Hairy Root Transformation.

Plants (Basel, Switzerland), 11(1): pii:plants11010051.

CRISPR/Cas-mediated genome editing is a powerful tool of plant functional genomics. Hairy root transformation is a rapid and convenient approach for obtaining transgenic roots. When combined, these techniques represent a fast and effective means of studying gene function. In this review, we outline the current state of the art reached by the combination of these approaches over seven years. Additionally, we discuss the origins of different Agrobacterium rhizogenes strains that are widely used for hairy root transformation; the components of CRISPR/Cas vectors, such as the promoters that drive Cas or gRNA expression, the types of Cas nuclease, and selectable and screenable markers; and the application of CRISPR/Cas genome editing in hairy roots. The modification of the already known vector pKSE401 with the addition of the rice translational enhancer OsMac3 and the gene encoding the fluorescent protein DsRed1 is also described.

RevDate: 2022-01-11

Kim EJ, Hong WJ, Kim YJ, et al (2021)

Transcriptome Analysis of Triple Mutant for OsMADS62, OsMADS63, and OsMADS68 Reveals the Downstream Regulatory Mechanism for Pollen Germination in Rice (Oryza sativa).

International journal of molecular sciences, 23(1): pii:ijms23010239.

The MADS (MCM1-AGAMOUS-DEFFICIENS-SRF) gene family has a preserved domain called MADS-box that regulates downstream gene expression as a transcriptional factor. Reports have revealed three MADS genes in rice, OsMADS62, OsMADS63, and OsMADS68, which exhibits preferential expression in mature rice pollen grains. To better understand the transcriptional regulation of pollen germination and tube growth in rice, we generated the loss-of-function homozygous mutant of these three OsMADS genes using the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9) system in wild-type backgrounds. Results showed that the triple knockout (KO) mutant showed a complete sterile phenotype without pollen germination. Next, to determine downstream candidate genes that are transcriptionally regulated by the three OsMADS genes during pollen development, we proceeded with RNA-seq analysis by sampling the mature anther of the mutant and wild-type. Two hundred and seventy-four upregulated and 658 downregulated genes with preferential expressions in the anthers were selected. Furthermore, downregulated genes possessed cell wall modification, clathrin coat assembly, and cellular cell wall organization features. We also selected downregulated genes predicted to be directly regulated by three OsMADS genes through the analyses for promoter sequences. Thus, this study provides a molecular background for understanding pollen germination and tube growth mediated by OsMADS62, OsMADS63, and OsMADS68 with mature pollen preferred expression.

RevDate: 2022-01-11
CmpDate: 2022-01-11

Hammouda OT, Wu MY, Kaul V, et al (2021)

In vivo identification and validation of novel potential predictors for human cardiovascular diseases.

PloS one, 16(12):e0261572.

Genetics crucially contributes to cardiovascular diseases (CVDs), the global leading cause of death. Since the majority of CVDs can be prevented by early intervention there is a high demand for the identification of predictive causative genes. While genome wide association studies (GWAS) correlate genes and CVDs after diagnosis and provide a valuable resource for such causative candidate genes, often preferentially those with previously known or suspected function are addressed further. To tackle the unaddressed blind spot of understudied genes, we particularly focused on the validation of human heart phenotype-associated GWAS candidates with little or no apparent connection to cardiac function. Building on the conservation of basic heart function and underlying genetics from fish to human we combined CRISPR/Cas9 genome editing of the orthologs of human GWAS candidates in isogenic medaka with automated high-throughput heart rate analysis. Our functional analyses of understudied human candidates uncovered a prominent fraction of heart rate associated genes from adult human patients impacting on the heart rate in embryonic medaka already in the injected generation. Following this pipeline, we identified 16 GWAS candidates with potential diagnostic and predictive power for human CVDs.

RevDate: 2022-01-11
CmpDate: 2022-01-11

Dong H, Zhang Y, Wang J, et al (2022)

Cas9-Based Local Enrichment and Genomics Sequence Revision of Megabase-Sized Shark IgNAR Loci.

Journal of immunology (Baltimore, Md. : 1950), 208(1):181-189.

The 0.8-Mb Ig new Ag receptor (IgNAR) region of the whitespotted bamboo shark (Chiloscyllium plagiosum) is incompletely assembled in Chr_44 of the reference genome. Here we used Cas9-assisted targeting of chromosome segments (CATCH) to enrich the 2 Mb region of the Chr_44 IgNAR loci and sequenced it by PacBio and next-generation sequencing. A fragment >3.13 Mb was isolated intact from the RBCs of sharks. The target was enriched 245.531-fold, and sequences had up to 94% coverage with a 255× mean depth. Compared with the previously published sequences, 20 holes were filled, with a total length of 3508 bp. In addition, we report five potential germline V alleles of IgNAR1 from six sharks that may belong to two clusters of the IgNAR. Our results provide a new method to research the germline of large Ig gene segments, as well as provide the enhanced bamboo shark IgNAR gene loci with fewer gaps.

RevDate: 2022-01-11
CmpDate: 2022-01-11

Klementieva N, Goliusova D, Krupinova J, et al (2021)

A Novel Isogenic Human Cell-Based System for MEN1 Syndrome Generated by CRISPR/Cas9 Genome Editing.

International journal of molecular sciences, 22(21):.

Multiple endocrine neoplasia type 1 (MEN1) is a rare tumor syndrome that manifests differently among various patients. Despite the mutations in the MEN1 gene that commonly predispose tumor development, there are no obvious phenotype-genotype correlations. The existing animal and in vitro models do not allow for studies of the molecular genetics of the disease in a human-specific context. We aimed to create a new human cell-based model, which would consider the variability in genetic or environmental factors that cause the complexity of MEN1 syndrome. Here, we generated patient-specific induced pluripotent stem cell lines carrying the mutation c.1252G>T, D418Y in the MEN1 gene. To reduce the genetically determined variability of the existing cellular models, we created an isogenic cell system by modifying the target allele through CRISPR/Cas9 editing with great specificity and efficiency. The high potential of these cell lines to differentiate into the endodermal lineage in defined conditions ensures the next steps in the development of more specialized cells that are commonly affected in MEN1 patients, such as parathyroid or pancreatic islet cells. We anticipate that this isogenic system will be broadly useful to comprehensively study MEN1 gene function across different contexts, including in vitro modeling of MEN1 syndrome.

RevDate: 2022-01-11
CmpDate: 2022-01-11

Yang X, Huang T, Wang T, et al (2021)

MAT2A-Mediated S-Adenosylmethionine Level in CD4+ T Cells Regulates HIV-1 Latent Infection.

Frontiers in immunology, 12:745784.

Antiretroviral drugs effectively halt HIV-1 replication and disease progression, however, due to the presence of a stable viral latent reservoir, the infection cannot be cured by antiretroviral drugs alone. Elucidating the molecular mechanisms underlying HIV-1 latent infection remains a critical hurdle that precludes the development of novel therapeutic strategies aiming for a potential functional cure. Cellular metabolism has been reported to affect HIV-1 replication in CD4+ T cells, but it remains largely unclear whether it is involved in the regulation of HIV-1 latency. Here, we performed a sub-pooled CRISPR library knockout screen targeting 1773 metabolic-related genes in a cell model of HIV-1 latent infection and found that Methionine Adenosyltransferase 2A (MAT2A) contributes to HIV-1 latency. MAT2A knockout enhanced the reactivation of latent HIV-1 while MAT2A overexpression did the opposite. Mechanistically, MAT2A modulates HIV-1 latency through S-Adenosylmethionine (SAM)-mediated one-carbon flux. MAT2A knockout resulted in a significant downregulation of DNA and histone methylation at the HIV-1 5'-LTR. Importantly, we found that the plasma level of SAM is positively correlated with HIV-1 DNA in PBMCs from ART-treated infected individuals, suggesting SAM could serve as a potential biomarker for the latent viral reservoir. Overall, this study reveals an important role of MAT2A-mediated one-carbon metabolism in regulating HIV-1 latency and provides a promising target for the development of new strategies for a functional cure of HIV-1.

RevDate: 2022-01-11
CmpDate: 2022-01-11

Chauvin L, Sevestre F, Lukan T, et al (2021)

Gene Editing in Potato Using CRISPR-Cas9 Technology.

Methods in molecular biology (Clifton, N.J.), 2354:331-351.

Genome editing in the cultivated potato (Solanum tuberosum), a vegetatively propagated and highly heterozygous species, constitutes a promising trail to directly improve traits into elite cultivars. With the recent and successful development of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system in eukaryotic cells, the plant science community has gained access to a powerful, inexpensive, and easy-to-use toolbox to target and inactivate/modify specific genes. The specificity and versatility of the CRISPR-Cas9 system rely on a variable 20 bp spacer sequence at the 5' end of a single-guide RNA (sgRNA), which directs the SpCas9 (Streptococcus pyogenes) nuclease to cut the target DNA at a precise locus with no or low off-target events. Using this system, we and other teams were able to knock out specific genes in potato through the error-prone non-homologous end-joining (NHEJ) DNA repair mechanism. In this chapter, we describe strategies to design and clone spacer sequences into CRISPR-SpCas9 plasmids. We show how these constructs can be used for Agrobacterium-mediated stable transformation or transient transfection of protoplasts, and we describe the optimization of these two delivery methods, as well as of the plant regeneration processes. Finally, the molecular screening and characterization of edited potato plants are also described, mainly relying on PCR-based methods such as high-resolution melt (HRM) analysis.

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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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CRISPR-Cas

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 @ gmail.com

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).

Timelines

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

Biographies

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 )