분자유전학실험실 (단국대학교 분자생물학과)



 이성욱 ( 2014-10-07 12:28:28 , Hit : 1422
 CRISPR/Cas9 Can Now Edit RNA, Not Just DNA

GEN News HighlightsMore »
Oct 6, 2014

The CRISPR/Cas9 system, a powerful tool for genome engineering and gene regulation, has been thought to be incapable of targeting RNA. This limitation, however, has been overcome by researchers led by CRISPR/Cas9 pioneer Jennifer Doudna, Ph.D., who holds joint appointments at the University of California, Berkeley and Howard Hughes Medical Institute.

These researchers reported that they have succeeded in programming CRISPR/Cas9 to recognize and cleave RNA at sequence-specific target sites. This advance, the researchers asserted, could enable direct RNA transcript detection, analysis, and manipulation.

The researchers’ RNA-targeting CRISPR/Cas9 complex, called RCas9, was detailed September 28 in Nature, in an article entitled, “Programmable RNA recognition and cleavage by CRISPR/Cas9.”
“Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide,” wrote the authors. “Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalyzed DNA cleavage.”

“While RNA interference has proven useful for manipulating gene regulation in certain organisms, there has been a strong motivation to develop an orthogonal nucleic-acid-based RNA-recognition system such as RCas9," Dr. Doudna said. “The molecular basis for RNA recognition by RCas9 is now clear and requires only the design and synthesis of a matching guide RNA and complementary PAMmer.”

The researchers envision a wide range of potential applications for RCas9. For example, an RCas9 tethered to a protein translation initiation factor and targeted to a specific mRNA could essentially act as a designer translation factor to “up-“ or “down-” regulate protein synthesis from that mRNA.

“Tethering RCas9 to beads could be used to isolate RNA or native RNA-protein complexes of interest from cells for downstream analysis or assays,” said Mitchell O’Connell, a researcher on Dr. Doudna’s team. “RCas9 fused to select protein domains could promote or exclude specific introns or exons, and RCas9 tethered to fluorescent proteins could be used to observe RNA localization and transport in living cells.”

“Using specially designed PAMmers, 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,” the authors of the Nature article concluded. “These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.”







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