Rapid and efficient generation of multigene-knockout THP-1 cells using a simple CRISPR-Cas9 protocol.

THP-1, a widely used human monocytic leukemia cell line, has been described as one of the hard-to-transfect suspension cell lines. Here, we present a rapid and efficient method for generating multiplex gene knockout cells from THP-1 using a simple co-transfection strategy based on the CRISPR-Cas9 system. In this method, THP-1 cells were transfected with a mixture of pX330-based sgRNA plasmids and a small amount of puromycin-resistance plasmid by electroporation, followed by transient selection with high-dose puromycin. This simple method resulted in efficient simultaneous knockout of multiple genes. For genes important for survival and/or cell proliferation, gene-knockout clones may be isolated by the limiting dilution method immediately after puromycin selection.•A simple co-transfection strategy to knock out multiple genes simultaneously in the human monocytic leukemia cell line, THP-1.•Transient selection with high-dose puromycin appeared to be effective for efficient knockout.•In general, knockout efficiency is satisfactory, and polyclonal knockout cells can be used without a cloning step unless the target gene plays a critical role in cell survival or proliferation.