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Dear Editor: The largest class of known human genetic diseases are caused by point mutations or single-nucleotide polymorphisms (SNPs). Base editors that can achieve targeted C-to-T conversions without generating DNA double-strand breaks, or requiring a donor template, represent significant advances in both disease modeling and gene therapy. However, the currently used base editor 3 (BE3), derived from the rAPOBEC1 (rA1)-nCas9 (Cas9 nickase, D10A) fusion, is limited by low editing efficiency in GC contexts, and by high bystander activity when more than one C is present in the editing window. Recently, human APOBEC3A (hA3A) was substituted for rA1 to produce new base editors (Fig. 1a). The editing window for hA3A-BEs was expanded to ~12 nt in human cells, and ~17 nt in plants and editing efficiency increased in GC contexts. An engineered hA3A variant, hA3AY130F, had a limited editing window, similar to that of BE3 (~6 nt in human cells). Another variant, hA3AN57G, showed a striking preference for the TCR (A/G) motif and a reduction in bystander mutations. In this study, we demonstrate the effectiveness of the hA3AnCas9 fusion and its associated variants (Y130F and N57G) to induce efficient and precise base editing in rabbits. We first tested hA3A-eBE in rabbit embryos at three target sites where GC (Tyr-1 and Tyr-3) or non-GC (Tyr2) contexts are contained in the editing window (Fig. 1b). As observed in human cells and plants, hA3A-eBE showed a significantly expanded base editing window (~20 nt), compared with that of BE3 (~8 nt), and a higher editing efficiency in the GC context at Tyr-1 and Tyr-3 in rabbit blastocysts (Fig. 1c; Supplementary Figs. S1–3). Moreover, the formation of by-products including indels and non-C-to-T conversions by hA3A-eBE was slightly reduced compared to that by BE3 (Supplementary Fig. S4). These results indicated that hA3A-eBE can induce efficient C-to-T base editing with a large editing window and, independently of context, it could increase genometargeting scope. Next, we examined the ability of hA3A-eBE-Y130F (narrowed editing window) to perform base editing in rabbit embryos. A total of six single guide RNAs (sgRNA) containing GC and non-GC contexts were selected to evaluate the editing efficiency of hA3A-eBE-Y130F (Fig. 1b). The results showed that the primary deamination window for hA3A-eBE-Y130F spanned 7 nt (from protospacer positions 3 to 9), and is similar to that of BE3 in rabbit embryos (Supplementary Figs. S1–3 and S5–8). The genomic DNA was edited at all target sites, with the efficiency of hA3A-eBE-Y130F being slightly higher than that of BE3 (81.11 ± 2.72% versus 75.33 ± 4.02%) in nonGC contexts (Fig. 1d, e), consistent with reported findings in human cells and mice. Within the editing window, in GC contexts, hA3A-eBE-Y130F induced significantly higher editing efficiencies than BE3 (63.00 ± 4.82% versus 28.44 ± 4.96%, p < 0.0001) (Fig. 1f, g). At all target sites, hA3A-eBE-Y130F-induced non-C-to-T conversions were significantly reduced (0.00 ± 0.00% versus 10.32 ± 3.02%, p < 0.01), but not indels, when compared with BE3 (Fig. 1h, i). The average frequency of by-products is relatively low using BE3, but in some samples, it reached 40–50%. This is consistent with the relatively high by-product frequency in individual mice and rabbit embryos using BE3 in previous studies. Overall, these results

Efficient and precise base editing in rabbits using human APOBEC3A-nCas9 fusions