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

## TL;DR

This study shows that the PE5max prime editor causes fewer unintended genetic changes compared to other variants, making it a safer option for gene editing.

## Contribution

The study introduces a comprehensive benchmarking framework combining PEM-seq and GOTI to evaluate the safety of prime editors.

## Key findings

- PE5max induces fewer large deletions and chromosomal translocations than PE3max.
- PE5max did not generate detectable off-target SNVs in mouse embryos using the GOTI assay.
- The study demonstrates PE5max has an improved specificity profile for safer therapeutic use.

## Abstract

What are the main findings?
PE5max induces far fewer large deletions and chromosomal translocations than PE3max, as measured by PEM-seq.In the GOTI assay using mouse embryos, PE5max was not observed to generate detectable genome-wide, sgRNA-dependent off-target SNVs within the detection limits of the assay.

PE5max induces far fewer large deletions and chromosomal translocations than PE3max, as measured by PEM-seq.

In the GOTI assay using mouse embryos, PE5max was not observed to generate detectable genome-wide, sgRNA-dependent off-target SNVs within the detection limits of the assay.

What are the implications of the main findings?
The study supports an improved specificity profile of PE5max relative to other PEmax systems in the assays used here, motivating further validation in additional cell types and in vivo settings.It provides a framework combining PEM-seq and GOTI for comprehensive, genome-wide safety evaluation of advanced genome editors.

The study supports an improved specificity profile of PE5max relative to other PEmax systems in the assays used here, motivating further validation in additional cell types and in vivo settings.

It provides a framework combining PEM-seq and GOTI for comprehensive, genome-wide safety evaluation of advanced genome editors.

Prime editing (PE) is a precise genome-editing technology that avoids double-strand breaks, holding great promise for clinical and agricultural applications. However, its genome-wide off-target effects are not fully understood, raising safety concerns. Here, we systematically compared the safety profiles of four prime editor variants (PE2max, PE3max, PE4max, and PE5max) using PEM-seq and RNA-seq. We further applied an ultra-sensitive method, Genome-wide Off-target analysis by Two-cell embryo Injection (GOTI), to assess PE5max. Our results show that PE5max did not produce detectable sgRNA-dependent off-target single-nucleotide variants (SNVs) in the GOTI assay and induced only limited large deletions and chromosomal translocations. Collectively, this side-by-side benchmarking under matched conditions demonstrates that PE5max achieves an improved specificity profile, with no detectable increase in genome-wide off-target SNVs, advancing its potential for safer therapeutic use.