Precise Insertion of AttB Sequences in Goat Genome Using Enhanced Prime Editor

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biotechnology

Background:

• Prime editing offers precise genomic modifications without DNA double-strand breaks.
• Current prime editing systems face limitations in efficiency due to target site, editing type, and primer binding site length.

Purpose of the Study:

• To enhance prime editing efficiency and broaden its application in gene insertion.
• To develop a more efficient prime editing system for genetic engineering in mammalian cells and embryos.

Main Methods:

• Incorporated an RNA motif into the 3' terminal of the pegRNA.
• Integrated all twin prime editor factors into a single plasmid.
• Applied the enhanced prime editor in goat cells and embryos for targeted sequence insertion.

Main Results:

• Achieved up to 3.58-fold and 2.19-fold enhancement in prime editing efficiency.
• Successfully inserted a 38 bp attB sequence into the Rosa26 and CCR5 loci in goat cells.
• Demonstrated editing efficiencies of 11.9% and 6.8% in goat embryos via microinjection.

Conclusions:

• The modified prime editing system exhibits improved editing and transfection efficiency.
• This enhanced system is suitable for inserting foreign sequences into primary cells and embryos.
• The study expands the potential of prime editing for transgenic animal development.