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

The largest class of known human genetic diseases are caused by point mutations or single-nucleotide polymorphisms (SNPs) 1 . 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 2 . 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 2 , 3 . 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 4 , 5 . An engineered hA3A variant, hA3A-Y130F, had a limited editing window, similar to that of BE3 (~6 nt in human cells) 4 , 6 . Another variant, hA3A-N57G, showed a striking preference for the TCR (A/G) motif and a reduction in bystander mutations 7 . In this study, we demonstrate the effectiveness of the hA3A-nCas9 fusion and its associated variants (Y130F and N57G) to induce efficient and precise base editing in rabbits. Fig. 1: hA3A-nCas9 fusions induce efficient and precise C-to-T base editing in vivo. The alternative text for this image may have been generated using AI. Full size image a Schematic representation of BE3 and hA3A-nCas9 fusion architecture. b Target-site sequences used in this study. Target sequence (black), sgRNA (bold), PAM region (green), target Cs (red, counting the PAM as positions 21–23). WT, wild-type. c Frequencies of single C-to-T conversions using BE3 and hA3A-eBE at three sites in rabbit embryos. GC contexts are indicated in bold on the x axis. d, f Comparison of C-to-T editing efficiency induced by BE3 and hA3A-eBE-Y130F in the target sites in non-GC context ( d ) or GC context ( f ). e, g Statistical analysis of the C-to-T editing frequency induced by BE3 and hA3A-eBE-Y130F in ( d ), ( f ), respectively. Forty-five target Cs for BE3 and 45 target Cs for hA3A-eBE-Y130F ( e ). Thirty-two target Cs for BE3 and 30 target Cs for hA3A-eBE-Y130F ( g ). h, i Statistical analysis of indels ( h ) or non-C-to-T conversions ( i ) induced by BE3 and hA3A-eBE-Y130F in rabbit embryos. Thirty-one embryos for BE3 and 30 embryos for hA3A-eBE-Y130F. j Frequencies of targeted single C-to-T conversions introduced by hA3A-eBE-Y130F and hA3A-eBE-N57G at three target sites in rabbit embryos. The TC contexts are indicated in red on the x axis. k The target sequence at the Tyr p. Q68Stop locus. The PAM and sgRNA target sequences are shown in green and black, respectively. Target mutation (red), bystander mutation (blue). l Representative Sanger sequencing chromatograms from a WT, and mutant rabbit (AT4). The red arrow indicates the substituted nucleotide. The relevant codon identities at the target sites are presented beneath the DNA sequence. m Comparison of the editing efficiency of single C-to-T conversions between BE3 and hA3A-eBE-N57G, at Tyr Q68Stop in F0 rabbit. The efficiency data of BE3 comes from our previous report 11 . n All five F0 rabbits exhibited a systemic albino phenotype. o H&E staining of skin from WT and Tyr mutant (AT4) rabbits. The red arrow highlights the melanin in the basal layer of the epidermis. Scale bars: 50 μm