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

• Published in: Nature biotechnology Vol. 31; no. 9; pp. 839 - 843
• Main Authors: Pattanayak, Vikram, Lin, Steven, Guilinger, John P, Ma, Enbo, Doudna, Jennifer A, Liu, David R
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.09.2013; Nature Publishing Group
• Subjects: 631/1647/1511; 631/337; 631/61; Agriculture; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bioinformatics; Biomedical Engineering/Biotechnology; Biomedicine; Biotechnology; Deoxyribonucleic acid; DNA; DNA - genetics; DNA - metabolism; DNA sequencing; Endonucleases - genetics; Endonucleases - metabolism; Enzymes; Gene expression; Genetic engineering; Genetic Engineering - methods; Genome; Genomics; Genomics - methods; HEK293 Cells; High-Throughput Nucleotide Sequencing - methods; Humans; letter; Life Sciences; Mutation; Nucleotide sequencing; Ribonucleic acid; RNA; RNA sequencing; RNA, Guide - genetics; Sequence Analysis, DNA - methods; Streptococcus pyogenes - enzymology; Streptococcus pyogenes - genetics; United States
• ISSN: 1087-0156; 1546-1696; 1546-1696
• DOI: 10.1038/nbt.2673

In vitro selection and high-throughput sequencing measure the sequence specificity of cleavage by gRNA-Cas9 complexes. 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.