Prevalence, formation, and function of “extraneous” CRISPR RNAs derived from the extra repeat in CRISPR arrays

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CRISPR-Cas systems in bacteria and archaea provide adaptive immunity against plasmids and phages. As part of adaptive immunity, small fragments of an invader’s genetic material are stored within a CRISPR array as spacers each located between fixed repeats. Each spacer-repeat pair then gives rise to a CRISPR RNA (crRNA) that directs the system’s effector nuclease to seek out and cleave nucleic-acid sequences matching the spacer. Because this spacer was derived from an invader, the crRNA helps confer resistance against this invader if it appears again.Each CRISPR array possessing the set of acquired spacers begins and ends with a repeat, leaving one repeat without a paired spacer. While the extra repeat is often necessary to acquire new spacers, it would also give rise to a crRNA with the spacer portion originating outside of the CRISPR array. This “extraneous” crRNA (ecrRNA) would be disconnected from adaptive immunity because the spacer portion was not derived from an invader, and it could interfere with crRNA biogenesis. Accordingly, we recently discovered different mechanisms by which CRISPR-Cas systems block formation of ecrRNAs. However, we observed instances in which these mechanisms were absent, potentially allowing natural ecrRNA formation. These observations raise the intriguing possibility that the produced ecrRNAs are functionally expressed and direct effector nucleases for purposes extending beyond adaptive immunity. Through this project, we will investigate the propensity of CRISPR-Cas systems to generate ecrRNAs and direct genome targeting by the effector nuclease. Our hypothesis is that functional ecrRNAs can be generated by different types of CRISPR-Cas systems. To investigate this hypothesis, we will explore three CRISPR sub-types (II-A, V-A, VI-B) in which we elucidated a distinct mechanism for blocking ecrRNA formation, with one objective devoted to each sub-type. We will apply complementary bioinformatics and experimental approaches to determine the extent ecrRNA formation and whether the resulting RNAs direct targeting by the effector nuclease. The proposed work represents an ongoing collaboration between the Weinberg and Beisel groups and combines their respective expertise in the bioinformatic prediction of functional RNAs and the experimental investigation of CRISPR biology. If successful, this project will establish the ecrRNA as a unique class of CRISPR-associated RNAs and reveal new modes by which CRISPR-Cas systems can enact functions beyond adaptive immunity.