Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair

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...

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Bibliographic Details
Published in:Nucleic acids research 2016-05, Vol.44 (9), p.e85-e85
Main Authors: He, Xiangjun, Tan, Chunlai, Wang, Feng, Wang, Yaofeng, Zhou, Rui, Cui, Dexuan, You, Wenxing, Zhao, Hui, Ren, Jianwei, Feng, Bo
Format: Article
Language:English
Subjects:
Cell Line, Tumor
Clustered Regularly Interspaced Short Palindromic Repeats - genetics
CRISPR-Cas Systems - genetics
DNA - genetics
DNA Breaks, Double-Stranded
DNA End-Joining Repair - genetics
Gene Editing - methods
Gene Knock-In Techniques
Genes, Reporter - genetics
Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) - genetics
Green Fluorescent Proteins - genetics
HCT116 Cells
HEK293 Cells
Human Embryonic Stem Cells - cytology
Humans
Methods Online
Recombinational DNA Repair - genetics
RNA, Guide, CRISPR-Cas Systems - genetics
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Summary: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.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkw064