An optimized prime editing system for efficient modification of the pig genome

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Animal Biotechnology

Background:

• Prime editing (PE) offers versatile gene editing but suffers from low efficiency in crucial agricultural species like pigs.
• Low PE efficiency limits its application in developing novel breeds and biomedical models in livestock.
• Porcine embryonic fibroblasts (PEFs) are a key model for studying gene editing in pigs.

Purpose of the Study:

• To enhance prime editing efficiency in porcine embryonic fibroblasts (PEFs).
• To identify strategies for optimizing both exogenous PE tools and endogenous pathways.
• To broaden the application of prime editing in agriculture and biomedicine.

Main Methods:

• Modified pegRNA by extending duplex length and altering thymine sequences to improve expression and cleavage.
• Inhibited SAMHD1, a deoxynucleoside triphosphate trihydrolase, using cephalosporin C zinc salt (CPC) to enhance reverse transcription.
• Administered histone deacetylase inhibitors (HDACis), including panobinostat, to increase transgene expression and editing efficiency.

Main Results:

• PegRNA modification significantly improved PE efficiency.
• CPC treatment increased PE efficiency up to 29-fold (4-fold on average).
• Panobinostat treatment yielded the highest efficiency increase, up to 122-fold (7-fold on average).
• Synergistic application of all three strategies further boosted PE efficiency in PEFs.

Conclusions:

• Developed novel strategies to significantly enhance prime editing efficiency in pigs.
• Optimized PE system broadens its potential applications in livestock breeding and biomedical research.
• Findings pave the way for advanced genetic engineering in important farm animals.