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Simultaneous generation of multi-gene knockouts in human cells.

ABSTRACT: 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.

SUBMITTER: Zhou Y

PROVIDER: S-EPMC5396285 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Simultaneous generation of multi-gene knockouts in human cells.

Zhou Yuexin Y Zhang Hongmin H Wei Wensheng W

FEBS letters 20161114 23

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 sp ...[more]

PMID: 27800615

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