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

项目摘要 CRISPR-Cas 12 a最近已成为一种强大的基因编辑工具，具有改善广泛遗传缺陷的巨大潜力。 通过基因疗法来治疗疾病Cas 12 a与Cas9一样，是一种可以被编程以切割 在特定的序列，它是高效率的基因组，但它是更好地针对AT丰富的序列和多个 基因同时然而，Cas 12 a的安全实施需要开发抑制剂 这可以实现调控并防止在非靶位点进行编辑。它还需要更好地了解 Cas 12 a生物学和细胞中的切割活性，这方面的数据很少。例如，Cas 12 a具有 已显示在结合其单链DNA后不加区别地切割单链DNA（即进行反式切割）。 在体外靶向DNA，但不知道这是否发生在细胞中。 该提案的长期目标是鉴定和开发Cas 12 a抑制剂，并确定Cas 12是否 反式切割发生在体内。利用生物信息学和体内分析，我们最近发现了第一个 三种蛋白质（acrVA 1 -3）抑制细菌和人类细胞中的Cas 12 a切割。这些蛋白质 在感染细菌的噬菌体（病毒）中编码，其中它们抑制Cas 12 a对噬菌体的切割。这些抑制剂 为Cas 12 a调控提供了有用的工具，但它们的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蛋白通过不同的 机制，这将使用各种体外和体内测定来阐明，这些测定确定它们对 Cas 12 a表达和靶DNA结合。接下来，我们将确定AcrVA蛋白，最佳抑制不同的 Cas 12 a变体通常用于基因编辑。这将通过诱变acrVA 1并选择 使用细菌选择筛选优化抑制剂，以及使用 生物信息学和体内抑制测定。最后，不加选择的Cas 12 a反式切割的存在， 将使用噬菌体感染实验测定其对acrVA 1 -3抑制的敏感性， 细菌总的来说，这项工作将阐明Cas 12 a的基本生物学，并将这些新的抑制剂开发成 这是一个可以调节Cas 12 a活性的强大工具。这样做，它将大大提高安全性和实用性， Cas 12 a在纠正遗传疾病中的作用这项工作将在加州大学旧金山分校进行，该大学拥有世界一流的设施 和一个高度智慧和协作的研究社区。它也将为我提供专业知识， 细菌噬菌体生物学，生物化学和基因编辑，我需要实现我的博士后培训目标， 开创了一项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.