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Although the efficiency and versatility of CRISPR‐Cas9 system has been greatly improved over conventional genome editing methods such as zinc finger or TALEN, it is still time‐consuming and labor‐intensive for screening knockout/knock‐in cell clones due to differences of the targeted location or efficacies of guide RNAs (gRNAs). Here, we adapted a targeted knock‐in strategy with CRISPR‐Cas9 system and characterized the efficiency for generating single or double knockout cell lines. Specifically, a homology‐arm based donor cassette consisting of genes encoding a fluorescence protein and antibiotic selection marker driven by a constitutive promoter was co‐transfected with a gRNA expressing unit. Based on FACS sorting and antibiotic drug selection, positive cell clones were confirmed by genotyping and at the protein expression level. The results indicated that more than 70% of analyzed clones identified by cell sorting and selection were successfully targeted in both single and double knockout experiments. The procedure takes less than three weeks to obtain knockout cell lines. We believe that this methodology could be applicable and versatile in generating knockout cell clones with high efficiency in most cell lines.

An efficient and precise method for generating knockout cell lines based on CRISPR‐Cas9 system