Fig. 3: Validation of selected genes at two endogenous sites in K562.

a, b The dCas9-BFP-KRAB and dox-inducible rA1-SaBE4(ΔUGI) expressing K562 cell line (Fig. 1b) was transduced with the C•G to T•A lentiviral reporter vector (Supplementary Fig. 2b), in which the BFP-targeting Sa-sgRNA spacer was replaced with either a HEK3- or RNF2-targeting spacer, and the CRISPRi Sp-sgRNA was targeted to the specific gene indicated. After a 3-day puromycin selection, doxycycline was added and rA1-SaBE4(ΔUGI) expression was induced for 7 days. The HEK3 or RNF2 locus (site of editing) was then sequenced with NGS to quantify editing efficiencies. Editing efficiencies of all outcomes at the two endogenous sites, HEK3 (a) and RNF2 (b), upon knockdown of selected genes are shown. Negative control (NC) samples are also shown, in which cells were treated identically using non-targeting sgRNAs. For the NC samples, the bars represent the mean obtained from 3 non-targeting sgRNAs, with n = 4 biological replicates each. For all other samples, the bars represent the mean of n = 2 (MSH6 and POLD1), 3 (LIG3), or 4 (UNG, MSH2, RFWD3, and ERCC4) biological replicates. The shaded regions represent the ranges that are within three STDs from the mean C•G to T•A and C•G to G•C editing efficiencies of the NC samples. c, d Cells were treated as described in (a, b), but with the BFP gene of the reporter construct replaced with cDNA encoding for the corresponding knocked-down gene. Shown are the fold-changes in editing efficiencies of all outcomes upon knockdown of selected genes at the two endogenous sites, HEK3 (c) and RNF2 (d), in comparison to the average editing efficiencies of 3 negative control samples without knockdown. The gene knockdown effects on all editing outcomes (solid color bars) at both sites were fully or partially rescued by the re-expression of the corresponding protein (striped bars). The bars represent the mean fold-changes for n = 4 and n = 3 biological replicates for the KD condition at HEK3 and RNF2 site, respectively, and n = 2 biological replicates for cDNA re-expression condition. KD, knockdown. Source data are provided as a Source Data file.