Figure 3

CRISPR/Cas9 templated gene editing analysis in mouse fibroblasts. (A) Overview of the sgRNA + 1 target site with sections of the sense and antisense (AS) ssODN that remove the c.828TAAA insertion and introduce a KpnI recognition site by a silent C > A substitution. Predicted cut site for Cas9 are indicated by “^˄” and “”, with “” indicating the strand that is nicked by Cas9D10A. Nucleotide numbers refer to the mutant c.828InsTAAA Fancf gene. (B) Overall gene editing efficiencies obtained in mouse fibroblasts depicted as general indel frequencies determined by TIDER (n = 3, one sided Student’s T-test, *p = 0.02). (C) Restriction fragment length polymorphism (RFLP) analysis by KpnI digest of the Fancf locus provides evidence for template-based repair after Cas9, sgRNA + 1 and ssODN transfection (n = 3, one sided Student’s T-test, *p < 0.03). (D) Mouse fibroblasts show enhanced clonal survival in the presence of 12.5 nM MMC after double-strand break formation by Cas9 and sgRNA + 1 independent of the orientation of the HDR template. No significant differences in relative survival were observed after Cas9 nickase activity (n = 3, one sided Student’s T-test, *p < 0.03). Error bars represent standard error, “M” = mock sgRNA.