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Engineering of high-precision base editors for site-specific single nucleotide replacement.
RNA-guided nucleases of the CRISPR/Cas type can be repurposed as programmable nucleotide deaminases to mediate targeted nucleotide substitutions. Such base editors have enormous potential in genome editing, gene therapy and precision breeding. However, current editors suffer from limited specificity in that they edit different and/or multiple bases within a larger sequence window. Using cytidine deaminase base editors that elicit C-to-T mutations, we show here that high editing precision can be achieved by engineering the connection between the deaminase domain and the Cas domain of the editor. By systematically testing different linker sequences and removing non-essential sequences from the deaminase, we obtain high-precision base editors with narrow activity windows that can selectively edit a single cytidine at a specific position with high accuracy and efficiency. These base editors will enable the use of genome editing in applications where single-nucleotide changes are required and off-target editing of adjacent nucleotides is not tolerable.
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Utilization of the CRISPR-Cas system for investigating tuberculosis drug resistance.
Drug-resistant tuberculosis(TB) presents a significant challenge to global efforts to control the disease. Addressing this issue requires a comprehensive understanding of drug resistance in Mycobacterium tuberculosis (M.tb). The clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (Cas) system are efficient genome-editing tools for various organisms and have facilitated new research directions regarding drug resistance in M.tb. This review summarizes recent progress in applying CRISPR/Cas technology to investigate drug resistance mechanisms in M.tuberculosis and discusses the potential of the CRISPR/Cas system as an integrated platform to transform research paradigms and enhance understanding of M.tuberculosis drug resistance mechanisms.
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Targeted mutagenesis in ryegrass (Lolium spp.) using the CRISPR/Cas9 system.
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Disabling Cas9 by an anti-CRISPR DNA mimic.
CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated gene editing in human cells. However, the mechanism of CRISPR-Cas9 inhibition is not known, and the potential applications for Cas9 inhibitor proteins in mammalian cells have not been fully established. We show that the anti-CRISPR protein AcrIIA4 binds only to assembled Cas9-single-guide RNA (sgRNA) complexes and not to Cas9 protein alone. A 3.9 Å resolution cryo-electron microscopy structure of the Cas9-sgRNA-AcrIIA4 complex revealed that the surface of AcrIIA4 is highly acidic and binds with a 1:1 stoichiometry to a region of Cas9 that normally engages the DNA protospacer adjacent motif. Consistent with this binding mode, order-of-addition experiments showed that AcrIIA4 interferes with DNA recognition but has no effect on preformed Cas9-sgRNA-DNA complexes. Timed delivery of AcrIIA4 into human cells as either protein or expression plasmid allows on-target Cas9-mediated gene editing while reducing off-target edits. These results provide a mechanistic understanding of AcrIIA4 function and demonstrate that inhibitors can modulate the extent and outcomes of Cas9-mediated gene editing.
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Ethical Considerations in Therapeutic Clinical Trials Involving Novel Human Germline-Editing Technology.
Much of the international community opposes editing the human germline. Yet, given enough experience to become better acquainted with strengths and limitations, prominent international figures are cautiously optimistic about using CRISPR-like novel technologies for clinical applications. Not only might such applications be morally (ethically) permissible, but clinical trials for therapeutic aims could be necessary. Here, we assess critical dimensions of early-phase trials deploying germline-editing technologies for "bench-to-bedside" translation. While assuming no overarching position favoring or opposing such research, our discussion primarily focuses on normative considerations. First, we evaluate the imperative of conducting trials to produce reliable, reproducible knowledge and advancement, if possible, for human diseases that are incurable and/or whose treatments are deficient. Second, we address complexities in assessing risk and potential-benefit profiles. Third, we review the moral foundations of trial participation through well-established and accepted bioethical principles: autonomy, nonmaleficence, beneficence, and distributive justice. Finally, we raise critical questions about the scope of regulatory authority and investigator and funder accountability for these applications that could have everlasting impacts.
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Advancing plant gene activation with CRISPR-Act3.0.
The pioneering plant genome-engineering tool CRISPR-Act3.0 is promising for precise gene activation, but faces challenges, such as unstable structures and inconsistent gene activation in polyploid genomes. Here, we propose strategic modifications to render CRISPR-Act3.0 more resilient and adaptable, especially in complex plant genomes, where conventional methods have struggled.
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CRISPR Screens in Toxicology Research: An Overview.
The use of genome editing tools is expanding our understanding of various human diseases by providing insight into gene-disease interactions. Despite the recognized role of toxicants in the development of human health issues and conditions, there is currently limited characterization of their mechanisms of action, and the application of CRISPR-based genome editing to the study of toxicants could help in the identification of novel gene-environment interactions. CRISPR-based functional screens enable identification of cellular mechanisms fundamental for response and susceptibility to a given toxicant. The aim of this review is to inform future directions in the application of CRISPR technologies in toxicological studies. We review and compare different types of CRISPR-based methods including pooled, anchored, combinatorial, and perturb-sequencing screens in vitro, in addition to pooled screenings in model organisms. © 2021 Wiley Periodicals LLC.
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Retroviral Vectors for Cancer Gene Therapy.
Advances in molecular technologies have led to the discovery of many disease-related genetic mutations as well as elucidation of aberrant gene and protein expression patterns in several human diseases, including cancer. This information has driven the development of novel therapeutic strategies, such as the utilization of small molecules to target specific cellular pathways and the use of retroviral vectors to retarget immune cells to recognize and eliminate tumor cells. Retroviral-mediated gene transfer has allowed efficient production of T cells engineered with chimeric antigen receptors (CARs), which have demonstrated marked success in the treatment of hematological malignancies. As a safety point, these modified cells can be outfitted with suicide genes. Customized gene editing tools, such as clustered regularly interspaced short palindromic repeats-CRISPR-associated nucleases (CRISPR-Cas9), zinc-finger nucleases (ZFNs), or TAL-effector nucleases (TALENs), may also be combined with retroviral delivery to specifically delete oncogenes, inactivate oncogenic signaling pathways, or deliver wild-type genes. Additionally, the feasibility of retroviral gene transfer strategies to protect the hematopoietic stem cells (HSC) from the dose-limiting toxic effects of chemotherapy and radiotherapy was demonstrated. While some of these approaches have yet to be translated into clinical application, the potential implications for improved cellular replacement therapies to enhance and/or support the current treatment modalities are enormous.
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Pairwise library screen systematically interrogates Staphylococcus aureus Cas9 specificity in human cells.
Therapeutic genome editing with Staphylococcus aureus Cas9 (SaCas9) requires a rigorous understanding of its potential off-target activity in the human genome. Here we report a high-throughput screening approach to measure SaCas9 genome editing variation in human cells across a large repertoire of 88,692 single guide RNAs (sgRNAs) paired with matched or mismatched target sites in a synthetic cassette. We incorporate randomized barcodes that enable whitelisting of correctly synthesized molecules for further downstream analysis, in order to circumvent the limitation of oligonucleotide synthesis errors. We find SaCas9 sgRNAs with 21-mer or 22-mer spacer sequences are generally more active, although high efficiency 20-mer spacers are markedly less tolerant of mismatches. Using this dataset, we developed an SaCas9 specificity model that performs robustly in ranking off-target sites. The barcoded pairwise library screen enabled high-fidelity recovery of guide-target relationships, providing a scalable framework for the investigation of CRISPR enzyme properties and general nucleic acid interactions.
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Efficient generation of goats with defined point mutation (I397V) in GDF9 through CRISPR/Cas9.
The recent emergence of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9 system has attracted significant attention for its potential to improve traits of agricultural importance. However, most applications in livestock species to date have depended on aberrant DNA repair to generate frameshifting indels. Whether this genomic engineering technique involving homology-dependent repair (HDR) can be used to introduce defined point mutations has been less explored. Previously, we reported a G→A point mutation (g.231A>G, p.Val397Ile) in the growth differentiation factor 9 (GDF9) gene that has a large effect on the litter size of cashmere goats. In the present study we report that by co-injecting synthesised RNAs and single-stranded oligo deoxynucleotide (ssODN) donor sequences into goat zygotes, we successfully introduced defined point mutations resulting in single amino acid substitutions in the proteins as expected. The efficiency of this precise single-nucleotide substitution in newborn kids was as high as 24% (4/17), indicating that ssODN-directed HDR via zygote injection is efficient at introducing point mutations in the goat genome. The findings of the present study further highlight the complex genome modifications facilitated by the CRISPR/Cas9 system, which is able to introduce defined point mutations. This represents a significant development for the improvement of reproduction traits in goats, as well as for validating the roles of specific nucleotides in functional genetic elements in large animals.
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