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

项目摘要/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 识别和切割具有序列特异性的噬菌体基因组。我们对这些机制的理解和 近年来，这些系统的应用有了很大的进步，然而，我们对 CRISPR-Cas与噬菌体相互作用的天然生理学缺乏。这份提案的重点是发现， 噬菌体对抗CRISPR-Cas免疫应答的特性和进化。我的实验室最近 发现单核细胞增生性李斯特菌噬菌体产生的抑制CRISPR-Cas9的“抗CRISPR”蛋白 通过不同的机制发挥作用。而其中三种蛋白质(AcrIIA2-4)直接与Cas9 RNA相互作用。 引导核酸酶AcrIIA1在没有这种相互作用的情况下发挥作用。而且，acrIIA1是最 迄今发现的广泛存在的抗CRISPR基因，由噬菌体、非噬菌体移动元件和核心编码 菲尔米特门的基因组。初步证据表明，这种蛋白质可以抑制 Cas9蛋白在细胞内的积累，提示对生物发生抑制的调节作用。不是这样的 之前已经描述过调节蛋白。AcrIIA1具有预测的螺旋-转角-螺旋结构域，该结构域 暗示了一种可能涉及核酸相互作用的机制。有趣的是，感染L. 单核细胞增多症并不只有一个抗CRISPR基因，它们通常编码AcrIIA1，除了至少 抑制蛋白之一(AcrIIA2-4)。这一明显的“多管齐下”的CRISPR的功能重要性- Cas9袭击事件未知。首先，我们将设计同基因噬菌体来确定多个抗体库的贡献 在裂解复制和溶源(噬菌体整合)过程中，CRISPR对噬菌体适合性的影响。第二，我们将确定 AcrIIA1是否与任何CRISPR-Cas9启动子元件或RNA转录本直接相互作用 审问它的作用机制。还将进行无偏见的交互评测，以完全捕获AcrIIA1 生物学。最后，鉴于acrIIA1同源基因的广泛存在，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，并确定功能所必需的残基。 初步分析表明，在Cispr-Cas9操纵子附近发现了acrIIA1同系物 乳杆菌，提示acrIIA1和内源性CRISPR-Cas9调节之间的功能连锁。 此外，我们将利用acrIIA1作为抗CRISPR的标记，以促进新的抗CRISPR的发现。这将是 为我们的最终目标做出贡献；识别所有被噬菌体抗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，作为 制定翻译后失活和限制非目标基因编辑的手段。总而言之，我建议 AcrIIA1是广泛存在于细菌和噬菌体中的CRISPR-Cas调节蛋白。我们将确定它的 作用、机制和多样性，这将极大地扩大我们对CRISPR-Cas生物学、噬菌体- 宿主相互作用，并为CRISPR-CAS应用贡献新的试剂。

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.