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

Abstract

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.