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: 9925050
• 负责人: Nicole D. Marino
• 金额: $ 6.53万
• 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 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9925050
• 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; 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; genetic testing; genome editing; improved; in vivo; inhibitor/antagonist; insight; novel; nuclease; off-target site; 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.

项目概要 CRISPR-Cas12a 最近成为一种强大的基因编辑工具，具有改善广泛疾病的巨大潜力。 通过基因治疗治疗多种疾病。 Cas12a 与 Cas9 一样，是一种可以编程切割的核酸酶 高效地定位特定序列的基因组，但更适合靶向富含 AT 的序列和多个 基因同时。然而，Cas12a 的安全实施需要开发抑制剂 这可以实现监管并防止在脱靶站点进行编辑。还需要加深对 Cas12a 生物学和细胞裂解活性，目前数据很少。例如，Cas12a 有 已被证明在与其结合后可以不加区别地切割单链 DNA（即进行反式切割） 体外靶向 DNA，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 反式裂解发生在体内。利用生物信息学和体内测定，我们最近发现了第一个 抑制细菌和人体细胞中 Cas12a 裂解的三种蛋白质 (acrVA1-3)。这些蛋白质是 在感染细菌的噬菌体（病毒）中编码，它们通过 Cas12a 抑制噬菌体裂解。这些抑制剂 将为 Cas12a 调控提供有用的工具，但其成功实施需要深入了解 他们的抑制机制。初步证据表明，每种 AcrVA 蛋白通过独特的方式发挥作用 机制，将通过各种体外和体内测定来阐明，以确定它们对 Cas12a 表达和靶 DNA 结合。接下来，我们将鉴定能够最佳抑制不同的 AcrVA 蛋白 Cas12a 变体常用于基因编辑。这将通过诱变 acrVA1 并选择来实现 使用细菌选择筛选以及探索天然 acrVA 多样性来优化抑制剂 生物信息学和体内抑制测定。最后，Cas12a 不加选择的反式切割的存在 体内及其对 acrVA1-3 抑制的敏感性将使用噬菌体感染实验来确定 细菌。总的来说，这项工作将阐明 Cas12a 的基本生物学原理，并将这些新型抑制剂开发成 可以调节 Cas12a 活性的强大工具。这样做，将显着提高安全性和实用性 Cas12a 纠正遗传性疾病。这项工作将在拥有世界一流设施的加州大学旧金山分校进行 以及一个高度智慧和协作的研究社区。它还将为我提供以下方面的专业知识 我需要实现我的博士后培训目标和细菌噬菌体生物学、生物化学和基因编辑 开创了细菌噬菌体反免疫的独立研究项目。