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'Class I and III Multi-subunit CRISPR-Cas Surveillance Complexes: Recognition, Cleavage, Autoimmunity and Inhibition’

“I 类和 III 类多亚基 CRISPR-Cas 监视复合物：识别、切割、自身免疫和抑制”

基本信息

批准号：
10360477
负责人：
DINSHAW J PATEL
金额：
$ 35.92万
依托单位：
SLOAN-KETTERING INST CAN RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-08 至 2024-03-31
项目状态：
已结题

项目摘要

Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign mobile genetic elements, such as invading phages and viruses. A central feature of this immune system is RNA- guided surveillance complexes capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner. The effector proteins are composed of either single-subunit Cas nucleases or the more prevalent multi-subunit CRISPR surveillance ribonucleoprotein complexes, together with either their intrinsic cis-acting or associated trans-acting helicase-nucleases. This application focuses on cryo-EM structural and biochemical (structure-guided interfacial mutational) studies to elucidate mechanistic insights related to dsDNA targeting by type I-F and ssRNA/ssDNA targeting by type III-A multi-subunit CRISPR systems, together with insights into cleavage mechanisms, as well as cleavage inhibition by phage-evolved anti-CRISPR proteins. Currently, the type III-A Csm is much less well characterized relative to its type-IIIB Cmr counterpart. We have recently solved cryo-EM based structures of crRNA-bound type III-A Csm (labeled CsmcrRNA) from T. onnurneus and its complexes with target RNA. In Aim 1 we propose to extend these studies to address structure-guided mechanistic issues related to the origins of autoimmunity suppression given that type III systems unlike type I lack a PAM sequence, to decipher the principles underlying target RNA-mediated transcription-coupled activation of ssDNA activity, as well as the generation of second messenger cyclic oligoadenyates, which in turn activate the nonspecific RNA degradation activity of trans-acting nuclease Csm6. We have recently solved cryo-EM based structures of crRNA-bound type I-F Csy complex (labeled CsycrRNA) from P. aeruginosa in the absence and presence of partial R-loop dsDNA and identified recognition principles and associated conformational transitions on ternary complex formation. Aim 2 focuses on extending this research to structures and conformational transitions of CsycrRNA on binding full R-loop dsDNA in the absence and presence of trans-acting helicase-nuclease Cas3. These efforts should address the principles underlying non-target DNA strand displacement and R-loop positioning for recognition and cleavage by Cas3. We have recently solved cryo-EM based structures of type I-F CsycrRNA with bound anti-CRISPR AcrF proteins 1, 2 and 10, thereby identifying alternate strategies utilized by AcrF suppressors for targeting and blocking different features of the dsDNA recognition machinery. Aim 3 focuses on a structure-based mechanistic understanding of the function of additional anti-CRISPR AcrF proteins 6, 7, 8 and 9 targeted to CsycrRNA, with the potential for identifying alternate anti-CRISPR approaches, including allosteric inhibition, for dsDNA cleavage suppression. To date, there have been no reports of anti-CRISPRs that target type III CRISPR-Cas systems, but should these be identified, we plan to extend our structural studies to these complexes towards characterization of the range of anti-CRISPR strategies for shutting down the type III CRISPR-Cas pathway.

原核细胞具有crispr介导的适应性免疫系统，保护它们免受外来移动