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CRISPR RNA-Dependent Binding and Cleavage of Endogenous RNAs by the Campylobacter jejuni Cas9

Author:
Dugar, Gaurav, Leenay, Ryan T., Eisenbart, Sara K., Bischler, Thorsten, Aul, Belinda U., Beisel, Chase L., Sharma, Cynthia M.
Source:
Molecular cell 2018 v.69 no.5 pp. 893-905.e7
ISSN:
1097-2765
Subject:
Campylobacter jejuni, DNA, binding sites, gene editing, messenger RNA, mutational analysis, nucleases
Abstract:
Cas9 nucleases naturally utilize CRISPR RNAs (crRNAs) to silence foreign double-stranded DNA. While recent work has shown that some Cas9 nucleases can also target RNA, RNA recognition has required nuclease modifications or accessory factors. Here, we show that the Campylobacter jejuni Cas9 (CjCas9) can bind and cleave complementary endogenous mRNAs in a crRNA-dependent manner. Approximately 100 transcripts co-immunoprecipitated with CjCas9 and generally can be subdivided through their base-pairing potential to the four crRNAs. A subset of these RNAs was cleaved around or within the predicted binding site. Mutational analyses revealed that RNA binding was crRNA and tracrRNA dependent and that target RNA cleavage required the CjCas9 HNH domain. We further observed that RNA cleavage was PAM independent, improved with greater complementarity between the crRNA and the RNA target, and was programmable in vitro. These findings suggest that C. jejuni Cas9 is a promiscuous nuclease that can coordinately target both DNA and RNA.
Agid:
5914893