Precise genomic deletions using paired prime editing

Area of Science:

• Molecular Biology
• Genomics
• Gene Editing Technologies

Background:

• Current clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 methods for genomic deletion are often inefficient and imprecise.
• Existing methods can result in unintended indels, large deletions, and complex genomic rearrangements.

Purpose of the Study:

• To introduce PRIME-Del, a novel prime editing-based system for precise genomic deletions.
• To evaluate the efficiency and precision of PRIME-Del compared to CRISPR-Cas9.

Main Methods:

• Utilizing a pair of prime editing sgRNAs (pegRNAs) targeting opposite DNA strands to program specific genomic deletions.
• Programming both nicking sites and repair outcomes for controlled deletion induction.
• Assessing deletion efficiency and precision for deletions up to 10kb.

Main Results:

• PRIME-Del demonstrates significantly higher precision in programming deletions compared to CRISPR-Cas9 and sgRNA pairs.
• Achieved editing efficiency for PRIME-Del ranges from 1-30%.
• PRIME-Del allows coupling genomic deletions with short insertions and deletions at non-protospacer-adjacent motif sites.

Conclusions:

• PRIME-Del offers a precise and flexible alternative for programming genomic deletions.
• The method can be enhanced by extended expression of prime editing components without compromising precision.
• PRIME-Del holds potential for applications including epitope tagging and programming genomic rearrangements.