Pattanayak, VikramLin, StevenGuilinger, John P.Ma, EnboDoudna, Jennifer A.Liu, David2014-04-112013Pattanayak, Vikram, Steven Lin, John P. Guilinger, Enbo Ma, Jennifer A. Doudna, and David R. Liu. 2013. “High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity.” Nature biotechnology 31 (9): 839-843. doi:10.1038/nbt.2673. http://dx.doi.org/10.1038/nbt.2673.1087-0156http://nrs.harvard.edu/urn-3:HUL.InstRepos:12064422The RNA-programmable Cas9 endonuclease cleaves double-stranded DNA at sites complementary to a 20-base-pair guide RNA. The Cas9 system has been used to modify genomes in multiple cells and organisms, demonstrating its potential as a facile genome-engineering tool. We used in vitro selection and high-throughput sequencing to determine the propensity of eight Cas9:guide RNA complexes to cleave each of 10^12 potential off-target DNA sequences. The selection results predicted five off-target sites in the human genome that were confirmed to undergo genome cleavage in HEK293T cells upon expression of one of two Cas9:guide RNA complexes. In contrast to previous models, our results show that Cas9:guide RNA specificity extends past a 7- to 12-base pair seed sequence. Our results also suggest a tradeoff between activity and specificity both in vitro and in cells as a shorter, less-active guide RNA is more specific then a longer, more-active guide RNA. High concentrations of Cas9:guide RNA complexes can cleave off-target sites containing mutations near or within the PAM that are not cleaved when enzyme concentrations are limiting.en-USJournal Article2014-04-1110.1038/nbt.2673