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

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.

RNA-Targeting CRISPR–Cas Systems and Their Applications

“…What differs this activity from the one described for F. novicida is that it does not utilize scaRNAs. It was shown that this activity is crRNA-, and tracrRNA-dependent, and that a site specific RNA cleavage is most probably performed by the Cas9 s HNH domain [58]. Neither PAM nor PFS sequences were shown to be required to regulate this activity [59].…”

“…Neither PAM nor PFS sequences were shown to be required to regulate this activity [59]. To date, this particular ssRNA targeting activity has been described for Staphylococcus aureus [60], Campylobacter jejuni [58], and Neisseria meningitides [59]. The exact role of the ssRNA targeting activity of type II CRISPR-Cas systems remains unknown.…”

The CRIPSR/Cas gene‐editing system—an immature but useful toolkit for experimental and clinical medicine

“…It can be used for eliminating pathogenic RNA molecules, adjusting aberrant mRNA expression and splicing. Nowadays, artificial RNA‐targeting Cas9s have programmable RNA modulating activity independent of Protospacer Adjacent Motif (PAM)‐presenting oligonucleotides (PAMmers) . CRISPR‐Cas13, another class 2 type VI CRISPR‐Cas system, specially targets RNA molecules and can be used for targeted gene knockdown in human cells .…”

In vivo epigenome editing and transcriptional modulation using CRISPR technology

“…The dCasRx splice effector was then packaged into the AAV vectors for tuning alternative splicing and correction of protein isoform ratios in postmitotic neurons of frontotemporal dementia (Konermann et al 2018). Although has not yet been demonstrated, it is also possible to package CjCas9 and SaCas9 into the AAV vectors for targeting and cleaving complementary endogenous mRNAs independent of a PAM (Dugar et al 2018; Strutt et al 2018). In this case, CjCas9 or SaCas9 nuclease alone is sufficient to alter the mRNA levels by directly targeting the RNA transcripts.…”