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 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.
Department of Chemistry & Chemical Biology and Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.
Vikram
V
Pattanayak
Steven
S
Lin
John P
JP
Guilinger
Enbo
E
Ma
Jennifer A
JA
Doudna
David R
DR
Liu
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D016678:Genome; D023281:Genomics / Q000379:methods; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D017422:Sequence Analysis, DNA / Q000379:methods*; D013297:Streptococcus pyogenes / Q000201:enzymology / Q000235:genetics; D058727:RNA, Small Untranslated
D001426:Bacterial Proteins; D004247:DNA; D004720:Endonucleases; D058727:RNA, Small Untranslated