Dugar, Gaurav; Leenay, Ryan T; Eisenbart, Sara K; Bischler, Thorsten; Aul, Belinda U; Beisel, Chase L; Sharma, Cynthia M
CRISPR RNA-Dependent Binding and Cleavage of Endogenous RNAs by the Campylobacter jejuni Cas9
Teil von
  • Molecular cell, 2018-03-01, Vol.69 (5), p.893-905.e7
Ort / Verlag
CAMBRIDGE: Elsevier Inc
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ScienceDirect Journals (5 years ago - present)
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. [Display omitted] •RIP-seq of the Campylobacter jejuni (Cj)Cas9 revealed numerous bound cellular RNAs•Most RNAs were co-purified with the CjCas9 in a crRNA-dependent manner•Some of the co-purified RNAs underwent cleavage dependent on the CjCas9 HNH domain•RNA cleavage by CjCas9 was programmable by an sgRNA in vitro CRISPR-Cas9 nucleases typically target double-stranded DNA targets. Dugar et al. show that one of the smallest Cas9 proteins (from Campylobacter jejuni) can also target endogenous RNAs. Targeting occurred through imperfect complementarity with native crRNA guides and cleavage by Cas9’s HNH domain.

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