Cas9 exo-endonuclease eliminates chromosomal translocations during genome editing

• Published in: Nature communications Vol. 13; no. 1; pp. 1204 - 14
• Main Authors: Yin, Jianhang, Lu, Rusen, Xin, Changchang, Wang, Yuhong, Ling, Xinyu, Li, Dong, Zhang, Weiwei, Liu, Mengzhu, Xie, Wutao, Kong, Lingyun, Si, Wen, Wei, Ping, Xiao, Bingbing, Lee, Hsiang-Ying, Liu, Tao, Hu, Jiazhi
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 08.03.2022; Nature Publishing Group; Nature Portfolio
• Subjects: 13/1; 13/100; 13/109; 13/31; 631/208/4041/3196; 631/337/1427/2122; Abnormalities; Antigens; Chimeric antigen receptors; Chromosome aberrations; Chromosome translocations; Cleavage; CRISPR; CRISPR-Associated Protein 9 - genetics; CRISPR-Cas Systems - genetics; DNA repair; Endonuclease; Endonucleases - genetics; Endonucleases - metabolism; Gene Editing; Genetic modification; Genome editing; Genomes; Humanities and Social Sciences; Humans; Lymphocytes; Lymphocytes T; multidisciplinary; Repair; RNA, Guide, CRISPR-Cas Systems - chemistry; RNA, Guide, CRISPR-Cas Systems - genetics; Science; Science (multidisciplinary); Translocation; Translocation, Genetic
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-022-28900-w

The mechanism underlying unwanted structural variations induced by CRISPR-Cas9 remains poorly understood, and no effective strategy is available to inhibit the generation of these byproducts. Here we find that the generation of a high level of translocations is dependent on repeated cleavage at the Cas9-targeting sites. Therefore, we employ a strategy in which Cas9 is fused with optimized TREX2 to generate Cas9TX, a Cas9 exo-endonuclease, which prevents perfect DNA repair and thereby avoids repeated cleavage. In comparison with CRISPR-Cas9, CRISPR-Cas9TX greatly suppressed translocation levels and enhanced the editing efficiency of single-site editing. The number of large deletions associated with Cas9TX was also reduced to very low level. The application of CRISPR-Cas9TX for multiplex gene editing in chimeric antigen receptor T cells nearly eliminated deleterious chromosomal translocations. We report the mechanism underlying translocations induced by Cas9, and propose a general strategy for reducing chromosomal abnormalities induced by CRISPR-RNA-guided endonucleases. Chromosomal structural variations induced by CRISPR/Cas hinder its application in clinics. Here, the authors fuse Cas9 with optimized TREX2 to generate Cas9TX, which can prevent perfect repair and inhibit repeated cleavage.