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Nature Biotechnology 2013, Vol. 31 (9) :839 -+ doi:10.1038/nbt.2673 <<-上一篇 下一篇 ->>
High-throughput profiling of off-target DNA cleavage reveals;RNA-programmed Cas9 nuclease specificity
Pattanayak, Vikram1,2;Guilinger, John P.1,2;Liu, David R.1,2;Lin, Steven3,4;Ma, Enbo3,4;Doudna, Jennifer A.3,4,5,6;
Pattanayak, Vikram1,2;Guilinger, John P.1,2;Liu, David R.1,2;Lin, Steven3,4;Ma, Enbo3,4;Doudna, Jennifer A.3,4,5,6;
Abstract: 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 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 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.
Keywords: ZINC-FINGER NUCLEASES GENE DISRUPTION CRISPR SYSTEMS CELLS ENDONUCLEASE SEQUENCE IMMUNITY TALENS
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