Uncovering the mechanism and role of a widespread anti-CRISPR-Cas9 protein

项目总结/摘要 细菌通过部署CRISPR-Cas免疫来预防病毒感染，CRISPR-Cas免疫的特点是RNA引导的核酸酶， 以序列特异性识别和切割噬菌体基因组。我们对这些机制的理解， 近年来，这些系统的应用取得了巨大的进展，然而，我们对这些系统的理解是， 缺乏CRISPR-Cas与大肠杆菌相互作用的天然生理学。这项提案的重点是发现， 噬菌体对CRISPR-Cas免疫的反应答的表征和进化。我的实验室最近 发现了由单核细胞增生李斯特菌产生的“抗CRISPR”蛋白， 通过不同的机制发挥作用。虽然其中三种蛋白质（AcrIIA 2 -4）直接与Cas9 RNA相互作用， 作为指导性核酸酶，AcrIIA 1在不存在这种相互作用的情况下发挥作用。此外，acrIIA 1是最 迄今为止发现的广泛的抗CRISPR基因，由噬菌体、非噬菌体移动的元件和核心编码 厚壁菌门的基因组初步证据表明，这种蛋白质抑制了 这表明Cas9蛋白在细胞中的积累，表明对生物发生抑制的调节作用。没有这样的 先前已经描述了调节蛋白。AcrIIA 1具有一个预测的螺旋-转角-螺旋结构域， 表明了一种可能涉及核酸相互作用的机制。有趣的是，感染L. 单核细胞增多症不仅仅具有一个抗CRISPR基因，它们通常编码AcrIIA 1，此外还至少编码 抑制蛋白（AcrIIA 2 -4）。这种明显的“多管齐下”CRISPR的功能重要性- Cas9攻击未知。首先，我们将设计等基因引物，以确定多个抗- 在裂解复制和溶原性（噬菌体整合）期间，CRISPR与噬菌体适应性。第二，我们将确定 AcrIIA 1是否与任何CRISPR-Cas9启动子元件或RNA转录物直接相互作用， 询问其作用机制。还将进行无偏倚相互作用分析，以完全捕获AcrIIA 1 生物学最后，考虑到acrIIA 1同源物的广泛性，我们将进行全面的生物信息学研究， 确定这个蛋白质超家族的进化起源，并确定功能的必需残基。 初步分析显示，在CRISPR-Cas9操纵子附近发现了acrIIA 1同源物， 这表明acrIIA 1和内源性CRISPR-Cas9调节之间的功能联系。 此外，我们将利用acrIIA 1作为抗CRISPR标记物，以促进新的抗CRISPR发现。这将 有助于我们的最终目标;确定所有被噬菌体抗CRISPR抑制的CRISPR-Cas系统 系统.此外，CRISPR-Cas9抑制剂为基因编辑工具箱提供了新的贡献， 这意味着制定翻译后失活并限制脱靶基因编辑。综上所述，我建议， AcrIIA 1是细菌和噬菌体拥有的一种广泛存在的CRISPR-Cas调节蛋白。我们将确定其 作用，机制和多样性，这将极大地扩展我们对CRISPR-Cas生物学，噬菌体- 宿主相互作用，并为CRISPR-Cas应用贡献新试剂。

PROJECT SUMMARY/ABSTRACT Bacteria prevent viral infection by deploying CRISPR-Cas immunity, which features RNA-guided nucleases that recognize and cleave phage genomes with sequence specificity. Our understanding of the mechanisms and applications for these systems has advanced dramatically in recent years, however, our appreciation for the natural physiology of CRISPR-Cas interactions with phages is lacking. This proposal focuses on the discovery, characterization and evolution of the phage counter-response to CRISPR-Cas immunity. My lab has recently discovered “anti-CRISPR” proteins produced by Listeria monocytogenes phages that inhibit CRISPR-Cas9 function through distinct mechanisms. While three of the proteins (AcrIIA2-4) interact directly with the Cas9 RNA- guided nuclease, AcrIIA1 functions in the absence of such an interaction. Moreover, acrIIA1 is the most widespread anti-CRISPR gene discovered to date, encoded by phages, non-phage mobile elements, and core genomes across the Firmicutes phylum. Preliminary evidence suggests that this protein represses the accumulation of Cas9 protein in the cell, suggesting a regulatory role towards biogenesis inhibition. No such regulatory protein has been previously described. AcrIIA1 possesses a predicted helix-turn-helix domain, which suggests a mechanism that may involve nucleic acid interactions. Interestingly, phages that infect L. monocytogenes do not possess just one anti-CRISPR gene, they often encode AcrIIA1, in addition to at least one of the inhibitor proteins (AcrIIA2-4). The functional importance of this apparent `multi-pronged' CRISPR- Cas9 attack is unknown. First, we will design isogenic phages to determine the contribution of multiple anti- CRISPRs to phage fitness, during lytic replication and lysogeny (phage integration). Second, we will determine whether AcrIIA1 makes direct interactions with any CRISPR-Cas9 promoter elements or RNA transcripts to interrogate its mechanism of action. Unbiased interaction profiling will also be conducted to fully capture AcrIIA1 biology. Lastly, given how widespread acrIIA1 homologs are, we will conduct comprehensive bioinformatics to determine the evolutionary origins of this protein superfamily and identify essential residues for function. Preliminary analyses have revealed an acrIIA1 homolog is found adjacent to a CRISPR-Cas9 operon in Lactobacillus, suggesting a functional linkage between acrIIA1 and endogenous CRISPR-Cas9 regulation. Additionally, we will utilize acrIIA1 as an anti-CRISPR marker to facilitate new anti-CRISPR discovery. This will contribute to our ultimate goal; identifying all CRISPR-Cas systems that are inhibited by phage anti-CRISPR systems. Additionally, CRISPR-Cas9 inhibitors provide new contributions to the gene editing toolbox, as a means to enact post-translational inactivation and limit off-target gene editing. Taken together, I propose that AcrIIA1 is a widespread CRISPR-Cas regulatory protein that bacteria and phage possess. We will determine its role, mechanism, and diverse reach, which will vastly expand our understanding of CRISPR-Cas biology, phage- host interactions, and contribute new reagents for CRISPR-Cas applications.