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

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 report11. 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？μ