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

揭示广泛存在的抗 CRISPR-Cas9 蛋白的机制和作用

基本信息

批准号：
10365999
负责人：
Joseph Bondy-Denomy
金额：
$ 33.92万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-04-09 至 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10365999
关键词：
Bacteria Bacterial Genes Bacteriophages Binding Biochemical Biogenesis Bioinformatics Biological Biological Assay Biology CRISPR/Cas technology Cells Clustered Regularly Interspaced Short Palindromic Repeats Data Elements Evolution Firmicutes Genes Genetic Genetic Markers Genome Genomics Goals Gram-Positive Bacteria Growth Guide RNA Helix-Turn-Helix Motifs Homologous Gene Immune Immune system Immunity Lactobacillus Light Listeria monocytogenes Lysogeny Lytic Methods Microbe Molecular Nucleic Acid Binding Nucleic Acids Operon Organism Pathway interactions Physiological Physiology Prophages Protein Family Proteins RNA Reagent Regulation Role Specificity Sum System Testing Transcript Virus Virus Diseases Work arms race design fitness gene discovery genetic regulatory protein genomic biomarker guided inquiry immune function in vivo inhibitor lytic replication microorganism novel novel marker nuclease pathogen prevent promoter response transcriptome sequencing

项目摘要

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.

项目概要/摘要细菌病毒通过部署 CRISPR-Cas 免疫来预防感染，该免疫具有 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。近年来，这些系统的应用取得了巨大的进步，但是，我们对缺乏 CRISPR-Cas 与噬菌体相互作用的自然生理学，该提案的重点是发现，我的实验室最近研究了噬菌体对 CRISPR-Cas 免疫反应的特征和进化。发现单核细胞增生李斯特氏菌噬菌体产生的“抗 CRISPR”蛋白可抑制 CRISPR-Cas9 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 是最重要的。迄今为止发现的广泛的抗 CRISPR 基因，由噬菌体、非噬菌体移动元件和核心编码整个厚壁菌门的基因组的初步证据表明，这种蛋白质抑制 Cas9 蛋白在细胞中的积累，表明对生物发生抑制具有调节作用。先前已描述过 AcrIIA1 具有预测的螺旋-转角-螺旋结构域。表明可能涉及核酸相互作用的机制。单核细胞增生李斯特菌不仅仅拥有一个抗 CRISPR 基因，它们通常编码 AcrIIA1，此外至少还编码 AcrIIA1 抑制剂蛋白之一 (AcrIIA2-4) 这种明显的“多管齐下”CRISPR-的功能重要性。 Cas9攻击未知。首先，我们将设计同基因噬菌体以确定多重抗的贡献。在裂解复制和溶原（噬菌体整合）过程中，CRISPR 对噬菌体的适应性。 AcrIIA1 是否与任何 CRISPR-Cas9 启动子元件或 RNA 转录物直接相互作用还将进行公正的相互作用分析以充分捕获 AcrIIA1。最后，考虑到 acrIIA1 同源物的广泛分布，我们将进行全面的生物信息学研究。确定该蛋白质超家族的进化起源并鉴定功能所必需的残基。初步分析表明，在 CRISPR-Cas9 操纵子附近发现了 acrIIA1 同源物乳酸菌，表明 acrIIA1 和内源 CRISPR-Cas9 调控之间存在功能联系。此外，我们将利用 acrIIA1 作为抗 CRISPR 标记，以促进新的抗 CRISPR 发现。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 系统此外，CRISPR-Cas9 抑制剂作为一种基因编辑工具，为基因编辑工具箱做出了新的贡献。意味着实施翻译后失活和限制脱靶基因编辑。综上所述，我建议。 AcrIIA1 是细菌和噬菌体所拥有的一种广泛存在的 CRISPR-Cas 调节蛋白，我们将确定它。作用、机制和多样化的影响范围，这将极大地扩展我们对 CRISPR-Cas 生物学、噬菌体的理解宿主相互作用，并为 CRISPR-Cas 应用提供新试剂。