Optimized Ribonucleoprotein Complexes Enhance Prime Editing Efficiency in Zebrafish.

Area of Science:

• Genetics
• Molecular Biology
• Aquatic Genomics

Background:

• Prime editing (PE) offers precise genome editing without double-strand DNA breaks (DSBs).
• Current PE applications in zebrafish show limited efficiency, hindering its use in this model organism.
• Enhancing PE efficiency is crucial for advancing functional gene studies and genetic breeding in zebrafish.

Purpose of the Study:

• To improve prime editing efficiency in zebrafish.
• To evaluate the efficacy of the PE7 system combined with La-accessible prime editing guide RNAs (pegRNAs).
• To demonstrate the utility of enhanced PE for generating specific mutations in zebrafish.

Main Methods:

• Utilized the PE7 prime editing system and La-accessible pegRNAs.
• Formed ribonucleoprotein (RNP) complexes by co-incubating PE7 protein with pegRNAs.
• Microinjected RNP complexes into zebrafish embryos to achieve targeted genome editing.

Main Results:

• Achieved up to 15.99% editing efficiency at target loci, a 6.81- to 11.46-fold increase over PE2.
• Observed 16.60% 6 bp insertions and 13.18% 10 bp deletions at the adgrf3b locus, a 3.13-fold increase over PE2.
• Successfully generated zebrafish with the tyr P302L mutation, showing reduced melanin, demonstrating PE's capability for specific trait modification.

Conclusions:

• PE7 significantly enhances prime editing efficiency in zebrafish.
• The combination of PE7 and La-accessible pegRNAs provides a powerful tool for precise genome editing in fish.
• This advancement facilitates functional gene studies and genetic breeding in aquatic species.