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

文献类型： 外文期刊

作者： Liu, Xiaoshuang 1 ; Gu, Dongfang 2 ; Zhang, Yiru 1 ; Jiang, Yingli 1 ; Xiao, Zhi 1 ; Xu, Rongfang 2 ; Qin, Ruiying 2 ; Li, Juan 2 ; Wei, Pengcheng 1 ;

作者机构： 1.Anhui Agr Univ, Coll Agron, Hefei 230036, Peoples R China

2.Anhui Acad Agr Sci, Rice Res Inst, Hefei 230031, Peoples R China

3.Anhui Agr Univ, Adv Acad, Res Ctr Biol Breeding Technol, Hefei 230036, Peoples R China

关键词： Prime editing; DNA mismatch repair; Rice; Fertility; Cre recombinase

期刊名称：GENOME BIOLOGY （ 影响因子：10.1； 五年影响因子：16.5 ）

ISSN： 1474-760X

年卷期： 2024 年 25 卷 1 期

页码：

收录情况： SCI

摘要： Background 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. Results 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 T-0 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. Conclusions 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.

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