Figure 4.

MRN recruitment improves editing of endogenous point mutation. (A). Site of the G690D ts mutation in BHK21 ts13 cells. The guide sequence targeting the mutation site is shown in bold, with the PAM site underlined. The ssODN used had 50bp homology arms. (B). Editing of the ts mutation in BHK21 ts13 is three to four times more efficient with MRN-recruiting constructs. BHK21 ts13 cells were transfected with the indicated Cas9/sgRNA expressing plasmids and the ssODN donor. Two days post-transfection, cells were replated, and transferred the next day to 39.5 °C. Two weeks later, colonies were stained with crystal violet and counted. Statistical analysis: Student’s t-test was performed, two-tailed, two-sample, unequal variance test. N = 7, *p < 0.02. (C,D). Reversion of TAF1 ts mutation in HEK293 is 2-fold more efficient with MRN-recruiting constructs. HEK293 TAF1 ts cells were transfected with the indicated Cas9/sgRNA-encoding plasmids and ssODN. Control non-targeting sgRNA and ssODN are listed in Supplementary Table S1. Cells were transfected and treated as in (B). C. Crystal violet-stained cells. D. Editing of HEK293 TAF1 ts by wt, U_N, U_C, and U_NLS Cas9. Statistical analysis as in (B) N = 14, * p < 0.011.