Discovery, Mechanism and Function of Type-V CRISPR-Cas Inhibitors

V型CRISPR-Cas抑制剂的发现、机制和功能

• 批准号: 9760566
• 负责人: Nicole D. Marino
• 金额: $ 6.12万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9760566
• 关键词: AT Rich Sequence; Affect; Bacteria; Bacteriophages; Binding; Biochemistry; Bioinformatics; Biological Assay; Biology; Cells; Cleaved cell; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; DNA; DNA Binding; Data; Development; Directed Molecular Evolution; Disease; Exhibits; Fluorescence; Genes; Genetic Diseases; Genetic Transcription; Genetic screening method; Genome; Goals; Guide RNA; Human; Immunity; Immunoprecipitation; In Vitro; Infection; Infection prevention; Lytic; Mediating; Methodology; Molecular Conformation; Mutagenesis; Mutation; Plaque Assay; Postdoctoral Fellow; Predisposition; Process; Proteins; RNA; Regulation; Reporter; Research; Resources; Safety; Single-Stranded DNA; Site; System; Testing; Variant; Virus; Work; base; ds-DNA; endonuclease; experimental study; gene therapy; genome editing; improved; in vivo; inhibitor/antagonist; insight; novel; nuclease; post-doctoral training; prevent; programs; protein function; reconstitution; tool; virtual

PROJECT SUMMARY CRISPR-Cas12a has recently emerged as a powerful gene editing tool with great potential to ameliorate wide- ranging diseases through gene therapy. Cas12a, like Cas9, is a nuclease that can be programmed to cut genomes at specific sequences with high efficiency, but it is better for targeting AT-rich sequences and multiple genes simultaneously. The safe implementation of Cas12a, however, requires the development of inhibitors that can enable regulation and prevent editing at off-target sites. It also requires improved understanding of Cas12a biology and cleavage activity in cells, for which a paucity of data exists. For example, Cas12a has been shown to indiscriminately cleave single-stranded DNA (i.e. perform trans-cleavage) after binding to its target DNA in vitro, but it is not known if this occurs in cells. The long-term objectives of this proposal are to identify and develop Cas12a inhibitors and determine if Cas12 trans-cleavage occurs in vivo. Using bioinformatics and in vivo assays, we have recently discovered the first three proteins (acrVA1-3) that inhibit Cas12a cleavage in bacteria and in human cells. These proteins are encoded in a phage (virus) infecting bacteria, where they inhibit phage cleavage by Cas12a. These inhibitors stand to provide useful tools for Cas12a regulation, but their successful implementation requires insight into their mechanisms of inhibition. Preliminary evidence suggests that each AcrVA protein functions by a distinct mechanism, which will be elucidated using a variety of in vitro and in vivo assays that determine their effect on Cas12a expression and target DNA binding. Next, we will identify AcrVA proteins that optimally inhibit different Cas12a variants commonly used in gene editing. This will be achieved by mutagenizing acrVA1 and selecting for optimized inhibitors using bacterial selection screens as well as by exploring natural acrVA diversity using bioinformatics and in vivo inhibition assays. Finally, the existence of indiscriminate Cas12a trans-cleavage in vivo and its susceptibility to inhibition by acrVA1-3 will be determined using phage infection experiments in bacteria. Overall, this work will illuminate fundamental Cas12a biology and develop these novel inhibitors into powerful tools that can regulate Cas12a activity. In doing so, it will significantly improve the safety and utility of Cas12a in correcting genetic disorders. This work will be performed at UCSF, which hosts world-class facilities and a highly intellectual and collaborative research community. It will also provide me with the expertise in bacterial-phage biology, biochemistry, and gene editing that I need to fulfill my postdoctoral training goals and pioneer an independent research program in bacterial-phage counter-immunity.

项目摘要 CRISPR-CAS12A最近成为了一种强大的基因编辑工具，具有改善广泛的潜力 通过基因疗法来疾病。 CAS12A（例如Cas9）是一种可以编程以切割的核酸酶 具有高效率的特定序列的基因组，但最好靶向富含富含的序列和多个 基因同时。但是，CAS12A的安全实施需要开发抑制剂 这可以实现调节并防止在脱离靶向地点进行编辑。它还需要提高对 CAS12A的生物学和细胞中的裂解活性，存在数据很少。例如，cas12a有 在与其结合后，已显示出不加选择的裂解单链DNA（即执行反裂解） 靶DNA在体外，但尚不清楚这是否发生在细胞中。 该提案的长期目标是识别和开发CAS12A抑制剂，并确定CAS12是否 反裂解发生在体内。使用生物信息学和体内测定，我们最近发现了第一个 三种蛋白质（ACRVA1-3）抑制细菌和人类细胞中Cas12a裂解的蛋白质。这些蛋白质是 在感染细菌的噬菌体（病毒）中编码，在其中抑制CAS12A的噬菌体裂解。这些抑制剂 为CAS12A法规提供有用的工具，但他们的成功实施需要深入了解 他们的抑制作用机制。初步证据表明，每种ACRVA蛋白通过一个独特的作用 机制将使用各种体外和体内测定法阐明，以确定其对 CAS12A表达和靶DNA结合。接下来，我们将确定最佳抑制不同不同的ACRVA蛋白 CAS12A变体通常用于基因编辑。这将通过诱变Acrva1和选择来实现 用于使用细菌选择筛选以及使用自然ACRVA多样性的优化抑制剂 生物信息学和体内抑制测定法。最后，存在不加区分的cas12a反式裂解 体内将使用噬菌体感染实验确定AcRVA1-3抑制作用的敏感性 细菌。总体而言，这项工作将阐明基本的CAS12A生物学，并将这些新颖的抑制剂发展为 可以调节CAS12A活动的强大工具。这样，它将大大提高 CAS12A纠正遗传疾病。这项工作将在UCSF上进行，该公司主持世界一流的设施 以及一个高度智力和协作的研究社区。它还将为我提供专业知识 我需要实现博士后培训目标和 先驱者在细菌含量的反免疫方面进行独立研究计划。