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

项目总结 CRISPR-Cas12a最近已成为一种强大的基因编辑工具，具有巨大的潜力来改善广泛的 通过基因疗法治疗各种疾病。Cas12a和Cas9一样，是一种可以被编程切割的核酸酶 基因组在特定序列上的效率很高，但它更适合于靶向AT丰富的序列和多个 基因同时存在。然而，Cas12a的安全实施需要开发抑制剂 这可以实现监管，并防止在目标之外的网站进行编辑。它还需要更好地理解 Cas12a在细胞中的生物学和切割活性，关于这方面的数据很少。例如，Cas12a有 被证明在与其结合后不分青红皂白地切割单链DNA(即执行反式切割) 在体外以DNA为靶标，但尚不清楚这种情况是否发生在细胞中。 该提案的长期目标是识别和开发Cas12a抑制剂，并确定Cas12 反式切割在体内发生。利用生物信息学和活体分析，我们最近发现了第一个 在细菌和人类细胞中抑制Cas12a切割的三种蛋白质(acrVA1-3)。这些蛋白质是 编码在感染细菌的噬菌体(病毒)中，在那里它们抑制Cas12a对噬菌体的切割。这些抑制剂 将为Cas12a监管提供有用的工具，但其成功实施需要深入了解 它们的抑制机制。初步证据表明，每种AcrVA蛋白都通过一种不同的 机制，这将通过各种体外和体内试验来阐明，以确定它们对 Cas12a的表达和靶DNA的结合。接下来，我们将确定AcrVA蛋白最能抑制不同的 通常用于基因编辑的Cas12a变体。这将通过诱变acrVA1和选择 对于使用细菌选择筛选的优化抑制剂以及通过使用 生物信息学和体内抑制试验。最后，存在不分青红皂白的Cas12a反式切割。 活体及其对acrVA1-3抑制的敏感性将通过噬菌体感染实验在 细菌。总之，这项工作将阐明Cas12a的基础生物学，并将这些新型抑制剂开发成 可以调节Cas12a活动的强大工具。通过这样做，它将显著提高安全性和实用性 CAS12A在纠正遗传疾病方面的作用。这项工作将在拥有世界级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 细菌噬菌体生物学、生物化学和基因编辑，我需要这些来完成我的博士后培训目标和 开创了细菌噬菌体抗免疫的独立研究计划。

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.