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. ! 1!

Public Health Relevance

Bacteria and archaea have developed a highly potent and adaptive CRISPR-Cas system that target and cleave foreign nucleic acids of invading viruses, phages and mobile genetic elements. The focus of this application is on the type I-F multi-subunit Csy complex that cleaves dsDNA and the type III-A multi-subunit Cmr complex that cleaves ssRNA and transcriptionally active ssDNA. We seek mechanistic insights into the divergent type I and III CRISPR-Cas systems from structure-function studies that define recognition events, conformational transitions between different states along the pathway, as well as cleavage of target nucleotides, thereby providing critical molecular insights into concepts necessary for site-specific and efficient genome engineering applications. !!

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function C Study Section (MSFC)
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Bender, Michael T
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Sloan-Kettering Institute for Cancer Research
New York
United States
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