Mechanisms and functions of endogenous RNA-targeting by CRISPR-Cas9 in Campylobacter jejuni

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The Type II CRISPR-Cas9 nucleases naturally utilize CRISPR RNAs (crRNAs) and tracrRNA to silence foreign double-stranded DNA. Besides their well-studied role in adaptive immunity, there is emerging evidence that CRISPR-Cas systems have roles beyond defense and impact on phenotypes such as bacterial virulence and group behavior. For example, deletion of Type II components affects virulence in diverse pathogens, such as Francisella novicida, Neisseria meningitidis or Campylobacter jejuni. While recent work has shown that some Cas9 nucleases can also target RNA, RNA recognition has required nuclease modifications or accessory factors. For example, F. novicida Cas9 uses an associated scaRNA and tracrRNA to repress an endogenous lipoprotein mRNA to affect virulence, although the exact mechanism of regulation remains unclear. Our RNA-seq analysis of multiple strains of the foodborne pathogen C. jejuni revealed a minimal crRNA biogenesis pathway for its abundantly transcribed Type II-C CRISPR-Cas system. Moreover, our unpublished RIP-seq (co-immunoprecipitation combined with RNA-seq) study revealed that C. jejuni Cas9 (CjCas9) uses its native crRNAs to bind and cleave complementary endogenous mRNAs. Approximately 100 transcripts co-purify with CjCas9 and generally can be subdivided through their base-pairing potential to the four crRNAs of strain NCTC11168. Several of these RNAs underwent cleavage close to the predicted binding site. Mutational analyses revealed RNA targeting was crRNA- and tracrRNA-dependent, and that RNA cleavage required the CjCas9 HNH domain. We further observed that RNA cleavage improved with greater complementarity between the crRNA and the RNA target, and was programmable in vitro. These findings suggest that CjCas9 is a promiscuous nuclease that can coordinately target both DNA and RNA.Here, we will further investigate the physiological roles and underlying molecular mechanisms of RNA targeting by CjCas9 to understand if it mediates endogenous gene regulation or is just a side effect of immune surveillance. First, RIP-seq of strains expressing different crRNA repertoires will reveal the extent of endogenous RNA targeting and reveal if there are separate crRNA-dependent and -independent regulons. Expression analyses in regulator mutants and under various conditions will show when CRISPR-Cas is regulated to provide insight into its function. RNA-seq and ribosome profiling of CRISPR-Cas mutants will identify global cleavage patterns and may identify targets of post-transcriptional control. We will then use genetics and biochemistry approaches to validate CjCas9 targets and study in detail the molecular mechanisms underlying regulation, including determinants for base-pairing with crRNAs, starting with selected candidate target mRNAs. Finally, we will determine if RNA targeting by CjCas9 regulates virulence. This will provide insight into the roles of CRISPR-Cas in bacterial gene regulation and pathogenicity.