Codon-optimized base editors enable efficient base substitution in Atlantic salmon

Area of Science:

• Genetics
• Molecular Biology
• Aquaculture Biotechnology

Background:

• Base editors (BEs) are powerful gene-editing tools but face challenges in non-model organisms due to codon usage bias.
• Existing BEs, like ABE8e and CBE4max-SpRY, are optimized for mammalian cells, limiting their efficacy in other species.

Purpose of the Study:

• To engineer and validate codon-optimized base editors (ss-ABE8e and ss-CBE4max-SpRY) for enhanced gene editing in Atlantic salmon (Salmo salar).
• To assess the efficiency and specificity of these optimized editors compared to their mammalian-derived counterparts.

Main Methods:

• Redesigned ABE8e and CBE4max-SpRY using Atlantic salmon preferred codons.
• Validated performance using in vitro reporter assays in salmon cells.
• Assessed in vivo editing efficiency via microinjection into fertilized salmon eggs.

Main Results:

• Codon-optimized BEs (ss-ABE8e, ss-CBE4max-SpRY) demonstrated efficient base substitutions in vitro and in vivo in Atlantic salmon.
• Engineered editors showed higher efficiency and lower bystander activity compared to original BEs.
• mRNA-encoded BEs exhibited lower editing patterns than plasmid-encoded BEs, likely due to mRNA stability.
• Successfully induced loss of protein function by mutating multiple loci to premature stop codons.

Conclusions:

• Codon optimization significantly improves base editor efficiency and specificity in Atlantic salmon.
• The developed salmon-specific base editors (ss-ABE8e, ss-CBE4max-SpRY) provide valuable tools for salmon gene editing and biotechnological applications.