Rapid generation of long, chemically modified pegRNAs for prime editing

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biochemistry

Background:

• Prime editing (PE) offers precise gene modification but faces limitations with current prime editing guide RNA (pegRNA) synthesis.
• Challenges in producing long pegRNAs (>125 nt) hinder optimal editing efficiencies, especially for ribonucleoprotein (RNP) and RNA delivery systems.

Purpose of the Study:

• To develop an efficient, rapid, and cost-effective method for synthesizing chemically modified long pegRNAs (L-pegRNAs) and long engineered pegRNAs (L-epegRNAs).
• To evaluate the performance of these novel RNAs in enhancing prime editing efficiencies across various cell types.

Main Methods:

• An optimized splint ligation approach was employed for the synthesis of L-pegRNAs (125-145 nt) and L-epegRNAs (170-190 nt).
• Production efficiency of approximately 90% was achieved for the synthesized long RNAs.
• Prime editing efficiencies were compared using L-epegRNA versus standard epegRNA with both RNP and RNA delivery systems, as well as plasmid-encoded PE.

Main Results:

• The developed method successfully generated high-quality L-pegRNA and L-epegRNA with desired chemical modifications.
• L-epegRNA demonstrated significantly enhanced prime editing efficiencies, with improvements up to tenfold for RNP delivery and several hundredfold for RNA delivery compared to standard epegRNA.
• RNP delivery utilizing L-epegRNA outperformed plasmid-encoded PE in most experimental comparisons.

Conclusions:

• The study presents a robust solution for producing high-quality, chemically modified long pegRNAs and epegRNAs.
• This advancement is crucial for overcoming current limitations in prime editing delivery and efficiency.
• The findings pave the way for broader applications of prime editing in therapeutic development and other research areas.