Dr Mitchell O’Connell, University of California, Berkeley, California, USA.
The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA–DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. CRISPR/Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms, but it has been thought to be incapable of targeting RNA. I will present our research showing that Cas9 is able to bind with high affinity to single-stranded RNA (ssRNA) targets in a guide-RNA programmable manner when an additional short DNA oligonucleotide containing a required motif known as the protospacer adjacent motif (PAM) is provided in trans. Furthermore, we have shown that these PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. Using specially designed PAMmers, we have shown that Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. We envisage that Cas9 will enable new ways of investigating and manipulating many aspects of RNA function, including targeted degradation of RNAs, isolation and identification of RNA-associated proteins, and in vivo imaging of RNAs.