Edited by Scott J. Hultgren, Washington University School of Medicine, St. Louis, MO, and approved April 8, 2015 (received for review November 24, 2014)
The clustered, regularly interspaced, short palindromic repeats associated endonuclease, Cas9, has quickly become a revolutionary tool in genome engineering. Utilizing small guiding RNAs, Cas9 can be targeted to specific DNA sequences of interest, where it catalyzes DNA cleavage. We now demonstrate that Cas9 from the Gram-negative bacterium Francisella novicida (FnCas9) can be reprogrammed to target a specific RNA substrate, the genome of the +ssRNA virus, hepatitis C virus, in eukaryotic cells. Further, this targeting results in inhibition of viral protein production. Overall, programmable Cas9-mediated viral RNA targeting likely represents one of myriad potential applications of FnCas9 in RNA targeting in eukaryotic cells.
Clustered, regularly interspaced, short palindromic repeats–CRISPR associated (CRISPR-Cas) systems are prokaryotic RNA-directed endonuclease machineries that act as an adaptive immune system against foreign genetic elements. Using small CRISPR RNAs that provide specificity, Cas proteins recognize and degrade nucleic acids. Our previous work demonstrated that the Cas9 endonuclease from Francisella novicida (FnCas9) is capable of targeting endogenous bacterial RNA. Here, we show that FnCas9 can be directed by an engineered RNA-targeting guide RNA to target and inhibit a human +ssRNA virus, hepatitis C virus, within eukaryotic cells. This work reveals a versatile and portable RNA-targeting system that can effectively function in eukaryotic cells and be programmed as an antiviral defense.
↵1A.A.P. and T.R.S. contributed equally to this work.
↵2Present address: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125.
Author contributions: A.A.P., T.R.S., H.K.R., A.G., and D.S.W. designed research; A.A.P., T.R.S., and H.K.R. performed research; A.A.P., T.R.S., H.K.R., A.G., and D.S.W. analyzed data; and A.A.P., T.R.S., H.K.R., A.G., and D.S.W. wrote the paper.
Conflict of interest statement: The authors have filed a related patent.
This article is a PNAS Direct Submission.
Data deposition: The sequence reported in this paper has been deposited in the NCBI BioProjects database (accession no. PRJNA281426).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1422340112/-/DCSupplemental.
Freely available online through the PNAS open access option.
