Constructing a C2C12 cell line that stably expresses Cas9 protein

【Objective】 To optimize the lentiviral packaging system and establish a mouse myoblast cell line（C2C12） that stably expressed Cas9 protein，providing a cellular model and theoretical reference for gene editing technology research. 【Method】 Using C2C12 cells as the research subject，control and experimental groups were set up. The dosage ratio of three plasmids was adjusted to optimize the lentiviral packaging system，and the plasmid expressing Cas9（lentiCas9-puro and lenti-Cas9-Blast）was packaged into lentivirus using the optimized system and infected C2C12 cells. C2C12 monoclonal cell lines with stable Cas9 expression were obtained by drug screening enrichment and monoclonal sorting. Genomic identification，cell immunofluorescence and sgRNA detection were conducted to verify the construction of the cell line. 【Result】 After optimization of the lentiviral packaging system，the plasmid transfection efficiency of the experimental group reached over 90%，which was extremely significantly higher than the control group（80%）（P<0.01）；the lentiviral titer of the experimental group increased to 1.8×10⁷ TU/mL，which was extremely significantly higher than the control group（6.2×106 TU/mL）（P<0.001）. PCR results showed that the C2C12-Cas9 stable cell line successfully amplified Cas9，puro，and BSD sequences，indicating that the exogenous Cas9 sequence was successfully integrated into the C2C12 cell genome，while wild-type C2C12 cells did not contain the Cas9 sequence. Immunofluorescence staining results indicated that the C2C12-Cas9 stable cell line could express Cas9 protein. T7E1 enzyme digestion and Sanger sequencing results indicated that the integrated Cas9 protein had cutting activity. 【Conclusion】 By adjusting the ratios of the three plasmids，the lentiviral packaging system is successfully optimized，improving plasmid transfection efficiency and lentiviral titer. C2C12 cell line that stably expresses Cas9 protein is successfully constructed，establishing a relatively mature system for constructing stable cell lines.