Fu, YanfangFoden, Jennifer A.Khayter, CydMaeder, Morgan L.Reyon, DeepakJoung, J. KeithSander, Jeffry D.2014-04-112013Fu, Yanfang, Jennifer A. Foden, Cyd Khayter, Morgan L. Maeder, Deepak Reyon, J. Keith Joung, and Jeffry D. Sander. 2013. “High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells.” Nature biotechnology 31 (9): 822-826. doi:10.1038/nbt.2623. http://dx.doi.org/10.1038/nbt.2623.1087-0156http://nrs.harvard.edu/urn-3:HUL.InstRepos:12064472CRISPR RNA-guided endonucleases (RGENs) have rapidly emerged as a facile and efficient platform for genome editing. Here, we use a human cell-based reporter assay to characterize off-target cleavage of Cas9-based RGENs. We find that single and double mismatches are tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface. We readily detected off-target alterations induced by four out of six RGENs targeted to endogenous loci in human cells by examination of partially mismatched sites. The off-target sites we identified harbor up to five mismatches and many are mutagenized with frequencies comparable to (or higher than) those observed at the intended on-target site. Our work demonstrates that RGENs are highly active even with imperfectly matched RNA-DNA interfaces in human cells, a finding that might confound their use in research and therapeutic applications.en-USJournal Article2014-04-1110.1038/nbt.2623