Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) systems are naturally occurring adaptive immune systems found in bacteria and archaea. There has been emerging evidence that Cas9 might have cellular roles beyond genome defence and can impact gene regulation or virulence. In our lab, we previously identified two distinct and unexpected modes of CjCas9 binding to endogenous RNAs. These are crRNA-dependent RNA targeting [1] and the discovery of non-canonical CRISPR RNAs (ncrRNAs), which are generated by novel mode of RNA-binding via duplex formation between trans-activating crRNA (tracrRNA) and cellular RNAs from outside the CRISPR-Cas9 locus [2]. Despite this novel discovery of ncrRNAs, it remained unclear whether formation of different cellular CjCas9 ribonucleoprotein (RNP) complexes play any role in endogenous gene regulation. Knowing that Cas9-mediated RNA cleavage requires crRNA-mRNA interactions in-vivo, we performed RIP-seq on C. jejuni mutant strains harbouring deletions of either individual crRNA, tracrRNA or the entire CRISPR locus. These analyses revealed endogenous RNAs that are exclusively enriched in these deletion strains, and those for which the binding to CjCas9 appeared to be independent of both crRNAs and tracrRNA. Further, using RIP-seq in complementation strains for wild-type and catalytically dead (dCas9) Cas9, we identify previously "hidden" RNA targets. We are currently examining these targets and exploring the possibility of complex tertiary structures on these RNAs aiding in their recognition by CjCas9, as exemplified by the presence of a unique triple-helix structure in native tracrRNA scaffold from C. jejuni. Overall, our findings provide new insights into cellular targets of CjCas9, uncovering its indelible impact on endogenous gene regulation, CRISPR biology and technologies.

References

[1] Dugar et al. (2018) Molecular Cell 69, 893–905.

[2] Jiao et al. (2021) Science 372, 6545.