• Xiangjun He, Chunlai Tan, Feng Wang, Yaofeng Wang, Rui Zhou, Dexuan Cui, Wenxing You, Hui Zhao, Jianwei Ren, and Bo Feng.
• Key Laboratory for Regenerative Medicine, Ministry of Education, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China CUHK-GIBH CAS Joint Research Laboratory on Stem Cell and Regenerative Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China.
• Nucleic Acids Res. 2016 May 19; 44 (9): e85.

AbstractCRISPR/Cas9-induced site-specific DNA double-strand breaks (DSBs) can be repaired by homology-directed repair (HDR) or non-homologous end joining (NHEJ) pathways. Extensive efforts have been made to knock-in exogenous DNA to a selected genomic locus in human cells; which, however, has focused on HDR-based strategies and was proven inefficient. Here, we report that NHEJ pathway mediates efficient rejoining of genome and plasmids following CRISPR/Cas9-induced DNA DSBs, and promotes high-efficiency DNA integration in various human cell types. With this homology-independent knock-in strategy, integration of a 4.6 kb promoterless ires-eGFP fragment into the GAPDH locus yielded up to 20% GFP+ cells in somatic LO2 cells, and 1.70% GFP+ cells in human embryonic stem cells (ESCs). Quantitative comparison further demonstrated that the NHEJ-based knock-in is more efficient than HDR-mediated gene targeting in all human cell types examined. These data support that CRISPR/Cas9-induced NHEJ provides a valuable new path for efficient genome editing in human ESCs and somatic cells.© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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