Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair
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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.
All Author(s) ListHe XJ, Tan CL, Wang F, Wang YF, Zhou R, Cui DX, You WX, Zhao H, Ren JW, Feng B
Journal nameNucleic Acids Research
Detailed descriptionTo ORKTS: doi:10.1093/nar/gkw064
Year2016
Month5
Day19
Volume Number44
Issue Number9
PublisherOXFORD UNIV PRESS
ISSN0305-1048
eISSN1362-4962
LanguagesEnglish-United Kingdom
Web of Science Subject CategoriesBiochemistry & Molecular Biology

Last updated on 2022-15-01 at 23:18