A simple, rapid, and efficient method for generating multigene-knockout culture cells by the CRISPR/Cas9 system.

We evaluated the efficacy of simultaneous multiple-gene knockout in human culture cells. By simple co-transfection of HeLa cells with a mixture of pX330-based targeting plasmids together with a puromycin resistance plasmid, followed by transient selection of puromycin-resistant cells, Cas9/single-guide RNA (sgRNA)-transduced polyclonal cell populations were selected and grown. Western blot analyses revealed co-transfection of up to seven targeting plasmids for p38α, p38β, JNK1, JNK2, Mnk1, ERK1, and mLST8 genes, drastically reduced protein expression of these genes in the polyclonal population. Analyses of a randomly isolated group of 25 clones revealed knockout efficiencies for the seven targeted genes ranging between 68% and 100%, and in six clones (24%), all targeted genes were disrupted. Deep sequencing analyses of the individual target sites revealed that, in most cases, Cas9/sgRNA-induced nonhomologous end joining resulted in deletion or insertion of only a few base pairs at the break points. These results demonstrate that simple co-transfection-based simultaneous targeting offers an easy, rapid, and efficient method to generate multiplex gene-knockout cell lines.