Fig. 4: Prevention of off-target effects by HDRobust and genetic end-joining repair inhibition.
From: Efficient high-precision homology-directed repair-dependent genome editing by HDRobust
a, Target site sequences of three different gRNAs that are predicted to be prone to off-target editing using CFD42 and MIT77 specificity scores. The sequences of the two off-targets with the highest CFD scores are shown below the on-target sites. Identical bases are given by dots. The CFD and MIT scores are in black frames. b, Genome editing efficiencies with Cas9, Cas9-HiFi or Cas9-HiFi with HDRobust at the on-target and top two CFD off-target sites in H9 hESCs without repair gene mutations. Independent biological replicates were performed (n = 3) and error bars show the s.e.m. c, Genome editing efficiencies at the on-target and top two CFD off-target sites by Cas9 RNP or Cas9-HiFi RNP in H9 hESCs without repair gene mutations, as well as with combinations of DNA-PKcs K3753R, Polθ V896* and RAD52 K152A/R153A/R156A (K/R152–156A). Independent biological replicates were performed (n = 3) and error bars show the s.e.m. d, Cell survival after editing with Cas9 RNP or Cas9-HiFi RNP in H9 hESCs without repair gene mutations, in combinations of DNA-PKcs K3753R, Polθ V896* and RAD52 K/R152–156A, as well as with HDRobust and Cas9-HiFi in wild-type cells. Cell survival was quantified by a fluorescence resazurin assay with respect to mock electroporation without editing. Independent biological replicates were performed (n = 3) and error bars show the s.e.m. Replicates are depicted by dots for HDR, NHEJ + MMEJ, or cell survival.
