title: Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair.
abstract: CRISPR/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.
authors: 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.
authors: Xiangjun
authors: X
authors: He
authors: 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.
authors: Chunlai
authors: C
authors: Tan
authors: 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.
authors: Feng
authors: F
authors: Wang
authors: 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 CUHK Shenzhen Research Institute, Shenzhen, 518057, China.
authors: Yaofeng
authors: Y
authors: Wang
authors: 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 Shenzhen Research Institute, Shenzhen, 518057, China.
authors: Rui
authors: R
authors: Zhou
authors: 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.
authors: Dexuan
authors: D
authors: Cui
authors: 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.
authors: Wenxing
authors: W
authors: You
authors: 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 CUHK Shenzhen Research Institute, Shenzhen, 518057, China.
authors: Hui
authors: H
authors: Zhao
authors: CUHK Shenzhen Research Institute, Shenzhen, 518057, China Department of Surgery, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China.
authors: Jianwei
authors: J
authors: Ren
authors: 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 CUHK Shenzhen Research Institute, Shenzhen, 518057, China fengbo@cuhk.edu.hk.
authors: Bo
authors: B
authors: Feng
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics*; D045744:Cell Line, Tumor; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D004247:DNA / Q000235:genetics; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair / Q000235:genetics*; D000072669:Gene Editing / Q000379:methods; D055879:Gene Knock-In Techniques; D017930:Genes, Reporter / Q000235:genetics*; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D045325:HCT116 Cells; D057809:HEK293 Cells; D000066449:Human Embryonic Stem Cells / Q000166:cytology; D006801:Humans; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D059767:Recombinational DNA Repair / Q000235:genetics*
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D049452:Green Fluorescent Proteins; D004247:DNA; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)