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Increasing the efficiency of CRISPR-Cas9-VQR precise genome editing in rice.
Clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) is a revolutionary technology that enables efficient genomic modification in many organisms. Currently, the wide use of Streptococcus pyogenes Cas9 (SpCas9) primarily recognizes sites harbouring a canonical NGG protospacer adjacent motif (PAM). The newly developed VQR (D1135V/R1335Q/T1337R) variant of Cas9 has been shown to cleave sites containing NGA PAM in rice, which greatly expanded the range of genome editing. However, the low editing efficiency of the VQR variant remains, which limits its wide application in genome editing. In this study, by modifying the single guide RNA (sgRNA) structure and strong endogenous promoters, we significantly increased the editing efficiency of the VQR variant. The modified CRISPR-Cas9-VQR system provides a robust toolbox for multiplex genome editing at sites containing noncanonical NGA PAM.
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Safety, Security, and Policy Considerations for Plant Genome Editing.
Genome editing with engineered nucleases (GEEN) is increasingly used as a tool for gene discovery and trait development in crops through generation of targeted changes in endogenous genes. The development of the CRISPR-Cas9 system (clustered regularly interspaced short palindromic repeats with associated Cas9 protein), in particular, has enabled widespread use of genome editing. Research to date has not comprehensively addressed genome-editing specificity and off-target mismatches that may result in unintended changes within plant genomes or the potential for gene drive initiation. Governance and regulatory considerations for bioengineered crops derived from using GEEN will require greater clarity as to target specificity, the potential for mismatched edits, unanticipated downstream effects of off-target mutations, and assurance that genome reagents do not occur in finished products. Since governance and regulatory decision making involves robust standards of evidence extending from the laboratory to the postcommercial marketplace, developers of genome-edited crops must anticipate significant engagement and investment to address questions of regulators and civil society.
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CRISPR Guide RNA Validation In Vitro.
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has been applied to edit genomes in a wide variety of model systems. Although this process can be quite efficient, editing at precise locations in the genome remains difficult without a suitable single guide RNA (sgRNA). We have developed a method for screening sgRNA function in vitro, using reagents that most zebrafish laboratories are already using. The results from our in vitro assay correlate with function in vivo in every sgRNA that we have examined so far. When combined with endonucleases with alternative protospacer adjacent motif site specificities and alternative sgRNAs, this method will streamline genome editing at almost any locus.
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Newer gene editing technologies toward HIV gene therapy.
Despite the great success of highly active antiretroviral therapy (HAART) in ameliorating the course of HIV infection, alternative therapeutic approaches are being pursued because of practical problems associated with life-long therapy. The eradication of HIV in the so-called "Berlin patient" who received a bone marrow transplant from a CCR5-negative donor has rekindled interest in genome engineering strategies to achieve the same effect. Precise gene editing within the cells is now a realistic possibility with recent advances in understanding the DNA repair mechanisms, DNA interaction with transcription factors and bacterial defense mechanisms. Within the past few years, four novel technologies have emerged that can be engineered for recognition of specific DNA target sequences to enable site-specific gene editing: Homing Endonuclease, ZFN, TALEN, and CRISPR/Cas9 system. The most recent CRISPR/Cas9 system uses a short stretch of complementary RNA bound to Cas9 nuclease to recognize and cleave target DNA, as opposed to the previous technologies that use DNA binding motifs of either zinc finger proteins or transcription activator-like effector molecules fused to an endonuclease to mediate sequence-specific DNA cleavage. Unlike RNA interference, which requires the continued presence of effector moieties to maintain gene silencing, the newer technologies allow permanent disruption of the targeted gene after a single treatment. Here, we review the applications, limitations and future prospects of novel gene-editing strategies for use as HIV therapy.
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Insights into gene manipulation techniques for Acari functional genomics.
Functional genomics is an essential tool for elucidating the structure and function of genes in any living organism. Here, we review the use of different gene manipulation techniques in functional genomics of Acari (mites and ticks). Some of these Acari species inflict severe economic losses to managed crops and health problems to humans, wild and domestic animals, but many also provide important ecosystem services worldwide. Currently, RNA interference (RNAi) is the leading gene expression manipulation tool followed by gene editing via the bacterial type II Clustered Regularly Interspaced Short Palindromic Repeats and associated protein 9 system (CRISPR-Cas9). Whilst RNAi, via siRNA, does not always lead to expected outcomes, the exploitations of the CRISPR systems in Acari are still in their infancy and are limited only to CRISP/Cas9 to date. In this review, we discuss the advantages and disadvantages of RNAi and CRISPR-Cas9 and the technical challenges associated with their exploitations. We also compare the biochemical machinery of RNAi and CRISPR-Cas9 technologies. We highlight some potential solutions for experimental optimization of each mechanism in gene function studies. The potential benefits of adopting various CRISPR-Cas9 systems for expanding on functional genomics experiments in Acari are also discussed.
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An Attenuated CRISPR-Cas System in Enterococcus faecalis Permits DNA Acquisition.
Antibiotic-resistant bacteria are critical public health concerns. Among the prime causative factors for the spread of antibiotic resistance is horizontal gene transfer (HGT). A useful model organism for investigating the relationship between HGT and antibiotic resistance is the opportunistic pathogen Enterococcus faecalis, since the species possesses highly conjugative plasmids that readily disseminate antibiotic resistance genes and virulence factors in nature. Unlike many commensal E. faecalis strains, the genomes of multidrug-resistant (MDR) E. faecalis clinical isolates are enriched for mobile genetic elements (MGEs) and lack clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) genome defense systems. CRISPR-Cas systems cleave foreign DNA in a programmable, sequence-specific manner and are disadvantageous for MGE-derived genome expansion. An unexplored facet of CRISPR biology in E. faecalis is that MGEs that are targeted by native CRISPR-Cas systems can be maintained transiently. Here, we investigate the basis for this "CRISPR tolerance." We observe that E. faecalis can maintain self-targeting constructs that direct Cas9 to cleave the chromosome, but at a fitness cost. Interestingly, DNA repair genes were not upregulated during self-targeting, but integrated prophages were strongly induced. We determined that low cas9 expression contributes to this transient nonlethality and used this knowledge to develop a robust CRISPR-assisted genome-editing scheme. Our results suggest that E. faecalis has maximized the potential for DNA acquisition by attenuating its CRISPR machinery, thereby facilitating the acquisition of potentially beneficial MGEs that may otherwise be restricted by genome defense.IMPORTANCE CRISPR-Cas has provided a powerful toolkit to manipulate bacteria, resulting in improved genetic manipulations and novel antimicrobials. These powerful applications rely on the premise that CRISPR-Cas chromosome targeting, which leads to double-stranded DNA breaks, is lethal. In this study, we show that chromosomal CRISPR targeting in Enterococcus faecalis is transiently nonlethal. We uncover novel phenotypes associated with this "CRISPR tolerance" and, after determining its genetic basis, develop a genome-editing platform in E. faecalis with negligible off-target effects. Our findings reveal a novel strategy exploited by a bacterial pathogen to cope with CRISPR-induced conflicts to more readily accept DNA, and our robust CRISPR editing platform will help simplify genetic modifications in this organism.
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Genome editing in rice and wheat using the CRISPR/Cas system.
Targeted genome editing nucleases, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), are powerful tools for understanding gene function and for developing valuable new traits in plants. The clustered regularly interspersed short palindromic repeats (CRISPR)/Cas system has recently emerged as an alternative nuclease-based method for efficient and versatile genome engineering. In this system, only the 20-nt targeting sequence within the single-guide RNA (sgRNA) needs to be changed to target different genes. The simplicity of the cloning strategy and the few limitations on potential target sites make the CRISPR/Cas system very appealing. Here we describe a stepwise protocol for the selection of target sites, as well as the design, construction, verification and use of sgRNAs for sequence-specific CRISPR/Cas-mediated mutagenesis and gene targeting in rice and wheat. The CRISPR/Cas system provides a straightforward method for rapid gene targeting within 1-2 weeks in protoplasts, and mutated rice plants can be generated within 13-17 weeks.
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Gene editing using CRISPR-Cas9 technology: potential implications in assisted reproduction.
PURPOSE OF REVIEW: This article reviews the mechanisms, advancements, and potential implications of clustered regularly interspaced short palindromic repeats-associated (CRISPR-Cas) gene editing technology, with a specific focus on its applications in reproductive biology and assisted reproduction. It aims to explore the benefits and challenges of integrating this revolutionary technology into clinical and research settings. RECENT FINDINGS: CRISPR-Cas9 is a transformative tool for precise genome editing, enabling targeted modifications through mechanisms like nonhomologous end joining (NHEJ) and homology-directed repair (HDR). Innovations such as Cas9 nickase and dCas9 systems have improved specificity and expanded applications, including gene activation, repression, and epigenetic modifications. In reproductive research, CRISPR has facilitated gene function studies, corrected genetic mutations in animal models, and demonstrated potential in addressing human infertility and hereditary disorders. Emerging applications include mitochondrial genome editing, population control of disease vectors via gene drives, and detailed analyses of epigenetic mechanisms. SUMMARY: CRISPR-Cas9 technology has revolutionized genetic engineering by enabling precise genome modifications. This article discusses its mechanisms, focusing on the repair pathways (NHEJ and HDR) and methods to mitigate off-target effects. In reproductive biology, CRISPR has advanced our understanding of fertility genes, allowed corrections of hereditary mutations, and opened avenues for novel therapeutic strategies. While its clinical application in human-assisted reproduction faces ethical and safety challenges, ongoing innovations hold promise for broader biomedical applications.
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CRISPR-Cas12a-Assisted Recombineering in Bacteria.
Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a (Cpf1) has emerged as an effective genome editing tool in many organisms. Here, we developed and optimized a CRISPR-Cas12a-assisted recombineering system to facilitate genetic manipulation in bacteria. Using this system, point mutations, deletions, insertions, and gene replacements can be easily generated on the chromosome or native plasmids in Escherichia coli, Yersinia pestis, and Mycobacterium smegmatis Because CRISPR-Cas12a-assisted recombineering does not require introduction of an antibiotic resistance gene into the chromosome to select for recombinants, it is an efficient approach for generating markerless and scarless mutations in bacteria.IMPORTANCE The CRISPR-Cas9 system has been widely used to facilitate genome editing in many bacteria. CRISPR-Cas12a (Cpf1), a new type of CRISPR-Cas system, allows efficient genome editing in bacteria when combined with recombineering. Cas12a and Cas9 recognize different target sites, which allows for more precise selection of the cleavage target and introduction of the desired mutation. In addition, CRISPR-Cas12a-assisted recombineering can be used for genetic manipulation of plasmids and plasmid curing. Finally, Cas12a-assisted recombineering in the generation of point mutations, deletions, insertions, and replacements in bacteria has been systematically analyzed. Taken together, our findings will guide efficient Cas12a-mediated genome editing in bacteria.
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Clinical applications of CRISPR-based genome editing and diagnostics.
Clustered regularly interspaced short palindromic repeats (CRISPR)-driven genome editing has rapidly transformed preclinical biomedical research by eliminating the underlying genetic basis of many diseases in model systems and facilitating the study of disease etiology. Translation to the clinic is under way, with announced or impending clinical trials utilizing ex vivo strategies for anticancer immunotherapy or correction of hemoglobinopathies. These exciting applications represent just a fraction of what is theoretically possible for this emerging technology, but many technical hurdles must be overcome before CRISPR-based genome editing technology can reach its full potential. One exciting recent development is the use of CRISPR systems for diagnostic detection of genetic sequences associated with pathogens or cancer. We review the biologic origins and functional mechanism of CRISPR systems and highlight several current and future clinical applications of genome editing.
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