Optimized prime editing efficiently generates heritable mutations in maize

Low efficiency is the main obstacle to using prime editing in maize(Zea mays).Recently,prime-editing efficiency was greatly improved in mammalian cells and rice(Oryza sativa)plants by engineering primeediting guide RNAs(pegRNAs),optimizing the prime editor(PE)protein,and manipulating cellular determinants of prime editing.In this study,we tested PEs optimized via these three strategies in maize.We demonstrated that the ePE5max system,composed of PEmax,epegRNAs(pegRNA-evopreQ.1),nicking single guide RNAs(sgRNAs),and MLH1dn,efficiently generated heritable mutations that conferred resistance to herbicides that inhibit 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS),acetolactate synthase(ALS),or acetyl CoA carboxylase(ACCase)activity.Collectively,we demonstrate that the ePE5max system has sufficient efficiency to generate heritable(homozygous or heterozygous)mutations in maize target genes and that the main obstacle to using PEs in maize has thus been removed.

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

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.