The protein Cas9, in complex with a guide RNA (gRNA) that targets a specific sequence, binds to and cleaves double-stranded DNA (dsDNA). Known as the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 system, this tool is touted for its ease of use and application across species. A team led by Jennifer Doudna at the University of California, Berkeley, has now extended the use of CRISPR-Cas9 to cleave single-stranded RNA (ssRNA) at specific target sites.

The key to the researchers' success lay with a short sequence called the protospacer-adjacent motif (PAM). Noting that PAM recognition is required for Cas9-gRNA to bind to DNA but that RNA lacks such a sequence, Doudna's team introduced a 'PAMmer'—an oligonucleotide containing a PAM—to anneal to the ssRNA and activate the cleavage process. They tested a number of PAMmer designs and discovered that specific cleavage occurred when several conditions were met. First, a PAMmer composed of deoxyribonucleotides resulted in cleavage, but a PAMmer of ribonucleotides did not. Second, a 5′ extension of the PAMmer prevented a mismatched gRNA from binding to the ssRNA: 2–8 nucleotides balanced specificity and binding efficiency. Third, a PAMmer and an ssRNA with an inexact base-pair match still yielded Cas9-gRNA cleavage while avoiding scission of PAM-less dsDNA incubated with this ssRNA, meaning that ssRNA could be specifically targeted even if genomic DNA were present.

Doudna's team put their system to the test by using it to isolate the GAPDH transcript from HeLa cell lysate. Initially they obtained two fragments of the transcript instead of a single whole; hypothesizing that cellular RNase H was the culprit, the team chemically modified the PAMmer to make it less susceptible to this unwanted cleavage. The modified PAMmer allowed them to isolate GAPDH intact without the need for affinity tags, cross-linking or probes.

The researchers suggest several uses for RNA recognition with CRISPR-Cas9. For example, cleavage with this system may be a helpful alternative when RNA interference cannot be used for RNA degradation. Using a catalytically inactive form of the nuclease, 'dRCas9', plus a marker, scientists could also potentially track RNA movement in cells. The programmable nature and high usability of this technology may prove a boon for researchers targeting or editing RNA, as its forerunner has done for DNA editing.