Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair.
Nucleic Acids Res
; 44(9): e85, 2016 05 19.
Article
em En
| MEDLINE
| ID: mdl-26850641
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Genes Reporter
/
Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)
/
Reparo do DNA por Junção de Extremidades
/
Reparo de DNA por Recombinação
/
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas
/
Sistemas CRISPR-Cas
Limite:
Humans
Idioma:
En
Revista:
Nucleic Acids Res
Ano de publicação:
2016
Tipo de documento:
Article