Extended Data Fig. 9: Optimization and characterization of CGBEfH/pH tools.
From: C-to-G editing generates double-strand breaks causing deletion, transversion and translocation
a, A schematic of the AID-CGBEfH1 construct is shown on top. Deletion frequencies at three sgRNA loci from three biological replicates are plotted at bottom. The same sgRNA loci are connected by a line. Mean ± SD is shown. b, Relative deletion frequency is shown as a heatmap for the AID-CGBEfH2 at sgFANCF locus. Deletion frequency was compared to that generated by AID-CGBE. c, Schematic illustration of C > G screening and protein evolution strategy. Validation of the mutations is shown on the right. Deletion and substitution frequencies are shown for each tool. Two biological replicates are shown for K83R/E165A/P185E/Q235K/Q235R. For other mutants, mean ± SD of 3 biological replicates is shown. For boxplots, centre line indicates the median; box limits represent the upper and lower quartiles, and the whiskers represent minimum to maximum values. d, Deletion and >1 bp insertion frequencies are plotted at the 7 sgRNA loci tested for the indicated tools. Mean ± SD of 3 biological replicates. Expression of CGBE tools is shown by Western blot. Representative blot is shown from 2 replicates. e, >1 bp insertion frequency is plotted at the 7 sgRNA loci tested for the indicated tools. Mean ± SD of 3 replicates. Expression of CGBE and HMCES is shown by Western blot. Representative blot is shown from 2 replicates. f, Deletion and C > G frequency are shown as mean ± SD of 3 biological replicates. g, C > G frequency are shown for synthetic ‘TCGA’ sgRNA sequences. Mean ± SD of 4 biological replicates. h, Deletion frequency is shown as mean ± SD of 3 replicates for the indicated tools. i, Numbers of normalized END-seq DSB ends are plotted along a 50 bp region of the sgEMX1 or sgFANCF locus. The orientation of DSB ends is indicated in blue (+) or red (-). j, Translocation junctions upon CGBE1 or CGBE1pH editing from the sgHBB bait. For Panels a, c, d, e, f, g and h, two-tailed Student’s paired t-test was applied. Source numerical data and unprocessed blots are available in source data.
