The three genes including ATP6V0A1，IL20RB，and STX10 were individually knocked out with CRISPR/Cas9 technology （previously established in author’s laboratory） in PK15-CD163-Cas9 cells and IPEC-J2-Cas9 cells to study the effects of three genes on the proliferation of porcine reproductive and respiratory syndrome virus （PRRSV） and porcine epidemic diarrhea virus （PEDV）.The cells with knocked-gene （PK15-CD163-Cas9-ATP6V0A1，PK15-CD163-Cas9-IL20RB and PK15-CD163-Cas9-STX10） were infected with PRRSV.The expression of the ORF7 of PRRSV was analyzed with fluorescence quantitative PCR to detect the proliferation of PRRSV.Meanwhile，the cells with knocked-gene （IPEC-J2-Cas9-ATP6V0A1，IPEC-J2-Cas9-IL20RB and IPEC-J2-Cas9-STX10） were infected with PEDV.The expression of N protein gene of PEDV and the proliferation of PEDV were detected.The results showed that the expression level of ORF7 in the cells with knocked-gene was significantly lower than that of the wildtype cells （PK15-CD163-Cas9-NC，control group）.The expression of N protein gene was significantly reduced in the cells with knocked-gene，compared with the wildtype cells （IPEC-J2-Cas9-NC，control group）.It is indicated that individually knocking out of the three genes significantly inhibits the proliferation of PRRSV and PEDV.ATP6V0A1，IL20RB，and STX10 can play important roles in the proliferation of PRRSV and PEDV.