Abstract: Objective To construct an in vitro cell model for elucidating the pathogenesis of peritoneal fibrosis by a knockout of PKCα gene in human peritoneal mesothelial cell line HMRSV5 with the technique of CRISPR/Cas9. Methods Three pairs of sgRNA sequences were designed for exon region of human PKCα gene. Two pairs of sequences were targeted at exon 4 and one pair exon 3. The oligomeric double-stranded DNA was synthesized by a primer synthetic company. The oligomeric double-stranded DNA was digested and ligated to eukaryotic expression vector GV393. The knockout efficiency of PKCα, i. e. off-target condition, was detected by using 293T cells as lentivirus packaging. Results Three pairs of sgRNA were successfully transfected into HMRSV5 cells by lentiviral expression vector. Real-time quantitative polymerase chain reaction(PCR) and Western blot revealed that PKCα was successfully knocked down in two pairs of sgRNA. The results of mismatch enzyme and gene sequencing indicated that PKCα genome DNA was effectively mutated and three off-target sites were consistent with the original reference sequence, excluding off-target condition. Conclusion The PKCα knockout HMRSV5 cells have been successfully constructed through the technique of CRISPR/Cas9. It may benefit further in vitro studies of related genes.