Simultaneous generation of multi‐gene knockouts in human cells

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.