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High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity


The 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 guide-RNA:Cas9 complexes to cleave each of 1012 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 guide-RNA:Cas9 complexes. In contrast to previous models, our results show that guide-RNA:Cas9 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 than a longer, more-active guide RNA. High concentrations of guide-RNA:Cas9 complexes can cleave off-target sites containing mutations near or within the PAM that are not cleaved when enzyme concentrations are limiting.

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Figure 1: In vitro selection overview.
Figure 2: In vitro selection results for Cas9:CLTA1 sgRNA.

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V.P., J.P.G. and D.R.L. were supported by DARPA HR0011-11-2-0003, DARPA N66001-12-C-4207, and the Howard Hughes Medical Institute. V.P. was supported by award no. T32GM007753 from the National Institute of General Medical Sciences. S.L. and J.A.D. were supported by the Howard Hughes Medical Institute (HHMI); E.M. was supported by US National Institutes of Health grant R01GM073794-05 to J.A.D.; J.A.D. and D.R.L. are HHMI Investigators.

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V.P., S.L., J.P.G. and E.M. performed the experiments, designed the research, analyzed the data and wrote the manuscript. J.A.D. and D.R.L. designed the research, analyzed the data and wrote the manuscript.

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Correspondence to Jennifer A Doudna or David R Liu.

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The authors have filed provisional patents related to this work.

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Supplementary Notes, Supplementary Figures 1–26, Supplementary Tables 2–8 and Supplementary Algorithms (PDF 16893 kb)

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Pattanayak, V., Lin, S., Guilinger, J. et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol 31, 839–843 (2013).

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