期刊
FEBS LETTERS
卷 590, 期 23, 页码 4343-4353出版社
WILEY-BLACKWELL
DOI: 10.1002/1873-3468.12469
关键词
CRISPR; knockout; nonhomologous end-joining
资金
- National Science Foundation of China [NSFC31430025, NSFC31170126, NSFC81471909]
- Beijing Advanced Innovation Center for Genomics at Peking University
- Peking-Tsinghua Center for Life Sciences
Genome-editing techniques enable the generation of gene knockouts in various mammalian cell lines. However, it remains technically challenging to completely disrupt a targeted gene using a canonical method in a timely manner. To improve the efficiency of producing reliable genomic modifications, we designed a method using a linear donor fragment containing a reporter system. Combined with a homologous recombination-independent knock-in strategy, we successfully enriched those cell clones that specifically carry the target gene mutations. We observed a much improved success rate when generating single- and multiple-gene knockouts in a one-step procedure using this special protocol coupled with the CRISPR/Cas9 system. This new approach further empowers the molecular biological study of genes and their functions.
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