Base editors (BEs) are promising gene-editing tools for correcting point mutations or generating new ones, broadly used in vitro and in model organisms, but their application in non-model animals remains limited. One of the major challenges is codon usage bias, as different organisms have preferences for specific codons to encode the same amino acid. Here, we redesigned the existing editors, ABE8e and CBE4max-SpRY, using salmon-preferred codons (designated ss-ABE8e and ss-CBE4max-SpRY), and validated their performance through a combination of in vitro reporter assays in Atlantic salmon (Salmo salar) cells and microinjection-based delivery in fertilized eggs. These engineered editors induced efficient base substitutions in vitro and in vivo. Compared with the original BEs (ABE8e and CBE4max-SpRY) derived from mammalian-cell models, our codon-optimized BEs exhibited higher efficiency but lower bystander (unintended on-target) activity by targeting the tyrosine gene in Atlantic salmon. Codon-optimized BEs encoded by mRNA showed fewer editing patterns than plasmid-encoded BEs, probably due to the limited lifespan of mRNA in cells. Furthermore, with these codon-optimized editors, we were able to mutate multiple loci, resulting in the complete loss of protein function in fish via premature stop codons. Our work demonstrates that codon optimization improves the efficiency of BEs in Atlantic salmon; ss-ABE8e and ss-CBEmax-SpRY increase the Atlantic salmon editing toolbox for biotechnological applications.
Wang et al. (Sun,) studied this question.