RNA-dependent RNA targeting by CRISPR-Cas9.

Steven C Strutt,Rachel M Torrez,Jennifer A Doudna,Emine Kaya,Oscar A Negrete

doi:10.7554/elife.32724

Abstract

Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo. We also demonstrate that Cas9 can direct PAM-independent repression of gene expression in bacteria. These results indicate that a subset of Cas9 enzymes have the ability to act on both DNA and RNA target sequences, and suggest the potential for use in programmable RNA targeting applications.

Highlights

Prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) systems provide immunity against plasmids and bacteriophage by using foreign DNA stored as CRISPR spacer sequences together with Cas nucleases to stop infection (Wright et al, 2016; Mohanraju et al, 2016)
No RNA cleavage was detected using S. pyogenes Cas9 (SpyCas9), which requires a PAMmer for efficient RNA cleavage in vitro (O’Connell et al, 2014), or using F. novicida Cas9 (FnoCas9)
Investigation of CRISPR-Cas9 has focused on its function as a double-stranded DNA endonuclease, while the ability of diverse homologs to cleave natural RNA substrates has remained unexplored

Summary

Introduction

Prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) systems provide immunity against plasmids and bacteriophage by using foreign DNA stored as CRISPR spacer sequences together with Cas nucleases to stop infection (Wright et al, 2016; Mohanraju et al, 2016) One such nuclease, Cas of the type II systems, employs a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) to target spacer-complementary regions (protospacers) on the foreign genetic element to guide double-stranded DNA cleavage (Jinek et al, 2012). Combining the crRNA and tracrRNA into a chimeric, single-guide RNA (sgRNA) simplified the system for widespread adoption as a versatile genome editing technology (Jinek et al, 2012) To date, both genetic and biochemical data support the conclusion that in vivo, Cas is exclusively a DNA-targeting enzyme. A different Cas homolog from Francisella novicida (FnoCas9) has been implicated in degradation of a specific mRNA

Methods

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Conclusion