Optimized prime editing efficiently generates glyphosate-resistant rice plants carrying homozygous TAP-IVS mutation in EPSPS  

作　　者：Yuanyuan Jiang Yiping Chai Dexin Qiao Junya Wang Cuiping Xin Wei Sun Zhenghong Cao Yu Zhang Yun Zhou Xue-Chen Wang Qi-Jun Chen 

机构地区：[1]State Key Laboratory of Plant Physiology and Biochemistry,College of Biological Sciences,China Agricultural University,Beijing 100193,China [2]Collaborative Innovation Center of Crop Stress Biology,Kaifeng,Henan Province 475004,China [3]Institute of Plant Stress Biology,School of Life Science,Henan University,Kaifeng 475004,China [4]Center for Crop Functional Genomics and Molecular Breeding,China Agricultural University,Beijing 100193,China

出　　处：《Molecular Plant》2022年第11期1646-1649,共4页分子植物（英文版）

基　　金：National Natural Science Foundation of China(grant nos.U19A2022 and 31872678);National Crop Breeding Fund(grant no.2016YFD0101804).

摘　　要：Dear Editor,Prime editing is a novel and universal CRISPR-Cas-derived precise genome-editing technology and has great potentials for applications in basic plant research and crop molecular breeding(Anzalone et al.,2019).Although low efficiency has restrained the original prime editors(PEs)from being used as a routine tool for precise genome editing in plants,an iterative update of the PEs is removing this obstacle(Lin et al.,2021;Xu et al.,2022).Recently,the Liu group reported three optimization strategies for improving prime editing efficiency(Chen et al.,2021;Nelson et al.,2022).The first strategy is based on engineered prime editing guide RNAs(epegRNAs),which were generated by incorporating structured RNA motifs to the 3′terminus of pegRNAs.This strategy enhances pegRNA stability and prevents degradation of the 3′extension(Nelson et al.,2022).The second strategy is based on the optimized PE2 protein(PEmax),which harbors a SpCas9 variant with increased nuclease activity,an additional nuclear localization signal(NLS)sequences,and a new linker between nCas9 and reverse transcriptase(Chen et al.,2021).The third strategy is based on inhibition of DNA mismatch repair(MMR)in cells(Chen et al.,2021).In this work,we tested the optimized PEs generated with these three strategies in rice,demonstrating that the optimized PEs greatly improved prime editing efficiency in rice.We named the two optimized PEs ePE3max and ePE5max:the former is comprised of the PEmax protein,an epegRNA with evopreQ1 appended to its 3′end,and a nicking sgRNA;the latter is comprised of the ePE3max system and a dominant negative OsMLH1 variant for inhibiting MMR.Using the two optimized PEs,we efficiently generated homozygous and heterozygous T173I,A174V,and P177S(TAP-IVS)mutation in EPSPS in rice,which lays a solid foundation for rice non-transgenic glyphosate-resistance breeding.

关 键 词：breeding precise EDITING 

分 类 号：S511[农业科学—作物学]