Targeting specificity of APOBEC-based cytosine base editor in human iPSCs determined by whole genome sequencing

Abstract

DNA base editors have enabled genome editing without generating DNA double strand breaks. The applications of this technology have been reported in a variety of animal and plant systems, however, their editing specificity in human stem cells has not been studied by unbiased genome-wide analysis. Here we investigate the fidelity of cytidine deaminase-mediated base editing in human induced pluripotent stem cells (iPSCs) by whole genome sequencing after sustained or transient base editor expression. While base-edited iPSC clones without significant off-target modifications are identified, this study also reveals the potential of APOBEC-based base editors in inducing unintended point mutations outside of likely in silico-predicted CRISPR-Cas9 off-targets. The majority of the off-target mutations are C:G->T:A transitions or C:G->G:C transversions enriched for the APOBEC mutagenesis signature. These results demonstrate that cytosine base editor-mediated editing may result in unintended genetic modifications with distinct patterns from that of the conventional CRISPR-Cas nucleases.

Directed Evolution of Adenine Base Editors with Increased Activity and Therapeutic Application

“…Importantly, all treated samples were confirmed to be bi-allelically edited at the on-target B2M locus, indicating receipt of active base editor or Cas9. Consistent with previous results from ABE7.10-d-treated animals in embryo injection mouse experiments 37,38,45,46 , we found no detectable increase (P-value = 0.911, one-sided Wilcoxon-Mann-Whitney U test, Supplementary Table 26 and Supplementary Table 27) in genome-wide A-to-G mutations upon treatment with ABE7.10-d (Fig. 4b, 4c and Supplementary Fig.…”

“…27). Concurrently, we identified a statistically significant increase in C-to-T mutations in BE4treated samples compared to untreated controls (P-value = 0.010, one-sided Wilcoxon-Mann-Whitney U test, Supplementary Table 26), consistent with previous reports 37,38,45,46 . We observed no statistically significant increase in A->G% mutation rates across the ABE8.17-m, ABE8.20-m or Cas9 groups when compared to untreated samples (P-value = 0.375, 0.643, and 0.27, respectively, one-sided Wilcoxon-Mann-Whitney U test, Fig.…”

“…Finally, we performed whole genome sequencing (WGS) to assess the degree to which ABE8s may induce genome-wide point mutations in a sgRNA-independent manner (referred to here as 'spurious deamination') as has been previously reported for CBEs, but not ABE7.10 or Cas9 37,38,45,46 . Accordingly, we transfected HEK293T cells with base editor-encoding mRNA and sgRNA targeting B2M (Site 21) and, after seventy-two hours of incubation, we used fluorescence activated cell sorting (FACS) to isolate B2M-negative single cells that had been successfully base edited.…”

Cytosine base editors with minimized unguided DNA and RNA off-target events and high on-target activity

“…4a). However, we identified a statistically significant increase in the ratio of C-to-T mutations following cellular treatment with BE4-rAPOBEC1 (as previously reported [6][7][8]21 ), BE4-PpAPOBEC1, and BE4-RrA3F F130L compared with untreated controls (P value = 0.018, 0.026, and 0.018, respectively, one-sided Wilcoxon-Mann-Whitney U test) ( Fig. 4a).…”

“…Whole genome sequencing (WGS) experiments were performed on base-editor-treated HEK293T cells grown by clonally expanding single cells ( Supplementary Fig. 15a) 21 . We compared the frequency of CBE-induced mutations in cells where base editors were delivered as either plasmid or mRNA.…”