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Simultaneous generation of multi‐gene knockouts in human cells
Author(s) -
Zhou Yuexin,
Zhang Hongmin,
Wei Wensheng
Publication year - 2016
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1002/1873-3468.12469
Subject(s) - gene knockout , crispr , genome editing , homologous recombination , gene , computational biology , gene targeting , cas9 , gene knockin , biology , genetics , genome , reporter gene , gene expression
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.