One-step base editing in multiple genes by direct embryo injection for pig trait improvement

Area of Science:

• Animal Genetics and Breeding
• Molecular Biology
• Biotechnology

Background:

• Developing elite pig breeders requires simultaneous acquisition of multiple beneficial genomic alleles.
• Cytidine base editors (CBEs) are effective tools for single C:G to T:A nucleotide substitutions.
• The efficacy of CBEs for multi-site editing in large animal zygotes was previously unverified.

Purpose of the Study:

• To determine the base-editing efficiency of four CBE variants in porcine cells and embryos.
• To verify if direct zygote microinjection of CBEs can generate pigs with multiple point mutations.
• To assess the impact of introduced mutations on economic traits and disease resistance in pigs.

Main Methods:

• Evaluated four Cytidine base editor (CBE) variants in porcine embryonic fibroblast cells and embryos.
• Utilized direct zygote microinjection of optimized CBE systems (hA3A-BE3-Y130F and hA3A-eBE-Y130F).
• Generated pigs with targeted mutations in CD163, myostatin (MSTN), and insulin-like growth factor-2 (IGF2).

Main Results:

• hA3A-BE3-Y130F and hA3A-eBE-Y130F demonstrated high editing efficiency with low toxicity in porcine embryos.
• Direct zygote microinjection successfully produced transgene-free pigs with multiple precise point mutations.
• Pigs exhibited disrupted CD163 and MSTN expression, increased IGF2 expression, improved growth, and enhanced disease resistance.

Conclusions:

• Optimized CBEs can effectively mediate simultaneous C-to-T substitutions at multiple loci in large animals.
• Direct embryo microinjection of CBEs enables rapid introduction of multiple beneficial mutations in pigs.
• This approach provides a novel strategy for comprehensively improving economic traits and producing elite pig breeders.