A benchmarked, high-efficiency prime editing platform for multiplexed dropout screening.

Area of Science:

• Genomics
• Molecular Biology
• Genetic Engineering

Background:

• Prime editing offers precise genomic modification but suffers from low and inconsistent editing efficiencies, limiting its use in high-throughput functional genomics.
• Developing efficient and reliable prime editing tools is crucial for advancing large-scale genetic variant analysis and functional studies.

Approach:

• Engineered a high-efficiency prime editing platform using optimized prime editing guide RNAs (epegRNAs) and target sequences, achieving a median 80% intended editing efficiency.
• Created a custom library of 240,000 epegRNAs to target over 17,000 codons with 175 distinct substitution types for functional interrogation of small nucleotide variants.
• Benchmarked the platform's specificity and efficiency in characterizing variants within essential genes, identifying phenotypes for nonsense and splice-site-disrupting synonymous mutations.

Key Points:

• Achieved high-efficiency substitution editing (80% median) with the developed prime editing platform.
• Successfully performed functional interrogation of over 17,000 codons using a large-scale epegRNA library.
• Identified functional consequences of specific genetic variants, including nonsense mutations and synonymous mutations affecting splice sites.

Conclusions:

• Established and validated a high-throughput prime editing approach for efficient and specific functional characterization of genetic variants.
• The developed platform facilitates rapid analysis of small substitution variants and their phenotypic effects in essential genes.
• This work advances the application of prime editing in functional genomics and variant interpretation.