Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors

• Published in: Nature biotechnology Vol. 33; no. 2; pp. 175 - 178
• Main Authors: Wang, Xiaoling, Wang, Yebo, Wu, Xiwei, Wang, Jinhui, Wang, Yingjia, Qiu, Zhaojun, Chang, Tammy, Huang, He, Lin, Ren-Jang, Yee, Jiing-Kuan
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.02.2015; Nature Publishing Group
• Subjects: 42; 45; 45/41; 631/1647/1511; 631/1647/1513/1967; 631/1647/1513/1967/2316; 631/1647/514/1948; Agriculture; Bioinformatics; Biomedical Engineering/Biotechnology; Biomedicine; Biotechnology; brief-communication; CRISPR-Cas Systems - genetics; Genetic engineering; Genetic research; Genetic Vectors; Genome, Human; Genomes; Genomics; HEK293 Cells; High-Throughput Nucleotide Sequencing; Humans; Integrases - genetics; Lentivirus; Lentivirus - enzymology; Lentivirus - genetics; Life Sciences; Methods; Nucleotide sequence; Ribonucleic acid; RNA; RNA Editing - genetics
• ISSN: 1087-0156; 1546-1696; 1546-1696
• DOI: 10.1038/nbt.3127

Off-target cleavage by CAS9 or TALEN genome editing tools is detected by integrase-defective lentiviral vectors. The utility of CRISPR-Cas9 and TALENs for genome editing may be compromised by their off-target activity. We show that integrase-defective lentiviral vectors (IDLVs) can detect such off-target cleavage with a frequency as low as 1%. In the case of Cas9, we find frequent off-target sites with a one-base bulge or up to 13 mismatches between the single guide RNA (sgRNA) and its genomic target, which refines sgRNA design.