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GENETICS Cleaving RNA with Cas9

CRISPR-Cas9 is not just for DNA any- the cleavage process. They tested a number chemically modified the PAMmer to make more: RNA targeting is also achievable. of PAMmer designs and discovered that spe- it less susceptible to this unwanted cleavage. The protein Cas9, in complex with a cific cleavage occurred when several condi- The modified PAMmer allowed them to guide RNA (gRNA) that targets a specific tions were met. First, a PAMmer composed isolate GAPDH intact without the need for sequence, binds to and cleaves double- of deoxyribonucleotides resulted in cleav- affinity tags, cross-linking or probes. stranded DNA (dsDNA). Known as the age, but a PAMmer of ribonucleotides did The researchers suggest several uses for clustered, regularly interspaced, short palin- not. Second, a 5ʹ extension of the PAMmer RNA recognition with CRISPR-Cas9. For dromic repeats (CRISPR)-Cas9 system, this prevented a mismatched gRNA from bind- example, cleavage with this system may be a tool is touted for its ease of use and applica- ing to the ssRNA: 2–8 nucleotides balanced helpful alternative when RNA interference tion across species. A team led by Jennifer specificity and binding efficiency. Third, cannot be used for RNA degradation. Using Doudna at the University of California, a PAMmer and an ssRNA with an inexact a catalytically inactive form of the nuclease, Berkeley, has now extended the use of base-pair match still yielded Cas9-gRNA ‘dRCas9’, plus a marker, scientists could also CRISPR-Cas9 to cleave single-stranded cleavage while avoiding scission of PAM-less potentially track RNA movement in cells. RNA (ssRNA) at specific target sites. dsDNA incubated with this ssRNA, mean- The programmable nature and high usabil- The key to the researchers’ success ing that ssRNA could be specifically targeted ity of this technology may prove a boon for lay with a short sequence called the even if genomic DNA were present. researchers targeting or editing RNA, as its protospacer-adjacent motif (PAM). Noting Doudna’s team put their system to the forerunner has done for DNA editing. that PAM recognition is required for Cas9- test by using it to isolate the GAPDH tran- Odelia Ghodsizadeh gRNA to bind to DNA but that RNA lacks script from HeLa cell lysate. Initially they RESEARCH PAPERS such a sequence, Doudna’s team introduced obtained two fragments of the transcript O’Connell, M.R. et al. Programmable RNA recognition a ‘PAMmer’—an oligonucleotide containing instead of a single whole; hypothesizing that and cleavage by CRISPR/Cas9. Nature a PAM—to anneal to the ssRNA and activate cellular RNase H was the culprit, the team doi:10.1038/nature13769 (28 September 2014). Nature America, Inc. All rights reserved. America, Inc. © 201 4 Nature npg

1090 | VOL.11 NO.11 | NOVEMBER 2014 | nature methods