Figure 6.

dCAS9 and Cpf1 impair NHEJ and induce genetic mutations. (A) Co-immunoprecipitation assay confirmed the interaction between dCAS9 and KU86. (B) Comet assay analysis of DNA damage in hESCs expressing dCAS9 or treated with doxorubicin. CTL, human fibroblasts with lentiviral empty vector were treated with 2 µg/mL doxycycline for three days; Doxy, Dox, Doxy + Dox, human fibroblasts with lentiviral CAS9 inducible expression vector were treated with 2 µg/mL doxycycline for 3 days or 0.5 µmol/L Dox for 2 h or 2 µg/mL doxycycline for three days + 0.5 µmol/L Dox for 2 h, respectively. Tail length was analyzed using Image J software. Data are represented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. (C) The expression of dCAS9 induces mutation of endogenous HPRT gene. WT, WT hESCs; CTL, CAS9, dCAS9, hESCs with empty expression vector, CAS9 inducible expression vector. Cells with dCAS9 inducible expression vector were treated with 2 µg/mL doxycycline for 3 days before HAT treatment. n = 3. Data are presented as mean value ± SD. **P < 0.01, ***P < 0.001. (D) The expression of Cpf1 increased the levels of γH2AX. (E) Cpf1 interacts with KU86 as confirmed by Co-immunoprecipitation. Protein extract of Flag-tagged Cpf1 was immunoprecipitated with anti-Flag antibody and the presence of Cpf1 and KU86 in the immunoprecipitate was examined by Western blot