# Conditional knockdown of OsMLH1 to improve plant prime editing systems without disturbing fertility in rice

## TL;DR

This paper introduces a method to improve plant genome editing efficiency in rice by temporarily inhibiting a DNA repair gene without affecting fertility.

## Contribution

A conditional RNAi knockdown of OsMLH1 in rice boosts prime editing efficiency while preserving fertility through an excisable system.

## Key findings

- Knockdown of OsMLH1 increased editing efficiency by 1.30- to 2.11-fold in rice cells.
- Up to 85.42% homozygous mutants were achieved in the T0 generation with high specificity.
- Conditional excision restored fertility without reducing editing efficiency in RNAi-free plants.

## Abstract

High-efficiency prime editing (PE) is desirable for precise genome manipulation. The activity of mammalian PE systems can be largely improved by inhibiting DNA mismatch repair by coexpressing a dominant-negative variant of MLH1. However, this strategy has not been widely used for PE optimization in plants, possibly because of its less conspicuous effects and inconsistent performance at different sites.

We show that direct RNAi knockdown of OsMLH1 in an ePE5c system increases the efficiency of our most recently updated PE tool by 1.30- to 2.11-fold in stably transformed rice cells, resulting in as many as 85.42% homozygous mutants in the T0 generation. The high specificity of ePE5c is revealed by whole-genome sequencing. To overcome the partial sterility induced by OsMLH1 knockdown of ePE5c, a conditional excision system is introduced to remove the RNAi module by Cre-mediated site-specific recombination. Using a simple approach of enriching excision events, we generate 100% RNAi module-free plants in the T0 generation. The increase in efficiency due to OsMLH1 knockdown is maintained in the excised plants, whose fertility is not impaired.

This study provides a safe and reliable plant PE optimization strategy for improving editing efficiency without disturbing plant development via transient MMR inhibition with an excisable RNAi module of MLH1.

The online version contains supplementary material available at 10.1186/s13059-024-03282-y.