Cas9 exo-endonuclease eliminates chromosomal translocations during genome editing

Jianhang Yin, Rusen Lu, Changchang Xin, Yuhong Wang, Xinyu Ling, Dong Li, Weiwei Zhang, Mengzhu Liu, Wutao Xie, Lingyun Kong, Wen Si, Ping Wei, Bingbing Xiao, Hsiang-Ying Lee, Tao Liu and Jiazhi Hu ()
Additional contact information
Jianhang Yin: Peking University
Rusen Lu: Peking University
Changchang Xin: Peking University
Yuhong Wang: Peking University
Xinyu Ling: Peking University
Dong Li: Peking University
Weiwei Zhang: Peking University
Mengzhu Liu: Peking University
Wutao Xie: Peking University
Lingyun Kong: Peking University
Wen Si: Peking University
Ping Wei: Peking University
Bingbing Xiao: Peking University First Hospital
Hsiang-Ying Lee: Peking University
Tao Liu: Peking University
Jiazhi Hu: Peking University

Nature Communications, 2022, vol. 13, issue 1, 1-14

Abstract: Abstract 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.

Date: 2022
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DOI: 10.1038/s41467-022-28900-w

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