Uncovering The Mechanism and Role of a Widespread Anti-CRISPR-cas9 Protein

<p>PROJECT SUMMARY/ABSTRACTBacteria prevent viral infection by deploying CRISPR-Cas immunity, which features RNA-guided nucleases thatrecognize and cleave phage genomes with sequence specificity. Our understanding of the mechanisms andapplications for these systems has advanced dramatically in recent years, however, our appreciation for thenatural 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 recentlydiscovered ?anti-CRISPR? proteins produced by Listeria monocytogenes phages that inhibit CRISPR-Cas9function 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 mostwidespread anti-CRISPR gene discovered to date, encoded by phages, non-phage mobile elements, and coregenomes across the Firmicutes phylum. Preliminary evidence suggests that this protein represses theaccumulation of Cas9 protein in the cell, suggesting a regulatory role towards biogenesis inhibition. No suchregulatory protein has been previously described. AcrIIA1 possesses a predicted helix-turn-helix domain, whichsuggests 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 leastone 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 determinewhether AcrIIA1 makes direct interactions with any CRISPR-Cas9 promoter elements or RNA transcripts tointerrogate its mechanism of action. Unbiased interaction profiling will also be conducted to fully capture AcrIIA1biology. Lastly, given how widespread acrIIA1 homologs are, we will conduct comprehensive bioinformatics todetermine 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 inLactobacillus, 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 willcontribute to our ultimate goal; identifying all CRISPR-Cas systems that are inhibited by phage anti-CRISPRsystems. Additionally, CRISPR-Cas9 inhibitors provide new contributions to the gene editing toolbox, as ameans to enact post-translational inactivation and limit off-target gene editing. Taken together, I propose thatAcrIIA1 is a widespread CRISPR-Cas regulatory protein that bacteria and phage possess. We will determine itsrole, 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.</p>