Prime Editing Exhibits Limited Genome-Wide Off-Target Effects in Cellular and Embryonic Gene Editing

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Prime editing (PE) is an advanced genome-editing technology.
• PE avoids double-strand breaks, offering therapeutic potential.
• Concerns exist regarding PE's genome-wide off-target effects.

Purpose of the Study:

• To systematically compare the safety profiles of four prime editor variants (PE2max, PE3max, PE4max, PE5max).
• To assess the genome-wide off-target effects of PE5max using the ultra-sensitive GOTI assay.
• To evaluate the specificity and safety of PE5max for potential therapeutic applications.

Main Methods:

• Comparative analysis of four prime editor variants (PE2max, PE3max, PE4max, PE5max).
• Utilized PEM-seq and RNA-seq for safety profiling.
• Employed Genome-wide Off-target analysis by Two-cell embryo Injection (GOTI) to assess PE5max specificity.

Main Results:

• PE5max demonstrated no detectable sgRNA-dependent off-target single-nucleotide variants (SNVs) via the GOTI assay.
• Limited large deletions and chromosomal translocations were observed with PE5max.
• Side-by-side benchmarking confirmed an improved specificity profile for PE5max.

Conclusions:

• PE5max exhibits enhanced specificity compared to other variants.
• The absence of detectable off-target SNVs supports PE5max's safety profile.
• PE5max represents a promising advancement for safer genome-editing therapeutics.