Fig. 5

Prime-editor-mediated multiplex precision genome editing in transgenic wheat plants. a Pipeline for prime editing of multiple endogenous genes by pB-CMPE-ePPEplus in wheat. Nine epegRNAs targeting eight endogenous genes were assembled in one Csy4 array. Of these genes, TaWTK3, TaACC-T2, TaSBEIIa, TaLOX2, TaDME, TaGW2, and TaGASR7 were targeted with a corresponding epegRNA, and with two epegRNAs respectively targeting copies of TaALS-T2 on the A and B/D subgenomes. BlpR, Bialaphos resistance. b Mutation frequencies of individual targeted genes in regenerated wheat plants. c Mutation frequencies of homoeologous genes in the A/B/D subgenomes for each targeted gene at each regenerated plant. Mutation efficiencies were examined by NGS with 5% threshold. Mutation frequency ≥ 70% was counted as homozygous mutation; mutation frequency ≥ 30 and < 70% was counted as heterozygous mutation; mutation frequency ≥ 5 and < 30% was counted as chimeric mutation; mutation frequency < 5% was counted as wild-type. When the main mutation type in a homozygous/ heterozygous/chimeric line contains undesired edits, it was counted as byproducts mutation. d Editing efficiencies of different mutation types of homoeologous genes in the A/B/D subgenomes for each targeted gene. e Frequencies of multiplex prime editing in regenerated wheat plants. f Ratio of simultaneous editing of different numbers of genes or genomic loci. n = 38 refers to the number of plants harboring two to eight genes mutated simultaneously. g Sanger sequencing chromatograms of the T₀-11 mutant harboring the desired prime edits in all eight genes. “*” indicates that the mutation type is chimeric. The protospacer-adjacent motif (PAM) sequence is highlighted in blue. The SNPs in different subgenomes are highlighted in green. The desired edits are highlighted in red