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

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 ce...