Fig. 4
From: Efficient and versatile multiplex prime editing in hexaploid wheat
Multiplex precision gene editing mediated by prime editor in wheat protoplasts. a Schematic diagrams of the constructs used for four distinct multiple pegRNAs/epegRNAs processing strategies. For the Pol III promoter-processing system, the wheat U3 (TaU3) and U6 (TaU6) promoters were used to drive expression of each pegRNA or epegRNA. For the tRNA-, ribozyme-, and Csy4-processing systems, a Pol II promoter, the Cestrum yellow leaf curling virus (CmYLCV) promoter, was employed to process polycistronic pegRNA/epegRNA transcripts. Csy4RS, Csy4 recognition site; HDV, hepatitis delta virus ribozyme; HH, hammerhead ribozyme; tRNA, 77 bp pre-tRNAGly genes. b Comparison of the four multiplex editing systems across four genes. Both pegRNAs and epegRNAs were used. c Overall mutation frequencies mediated by the Pol III promoter-, tRNA-, ribozyme-, and Csy4-processing systems. P values were obtained using the two-tailed Student’s t test. *P < 0.05, **P < 0.01. d Multiplexed mutagenesis of four, five, six, eight, nine, and ten simultaneously edited genes using CMPE-PPE, CMPE-ePPE, and CMPE-ePPEplus. CMPE, Csy4-mediated multiplex prime editing. e Frequencies of each targeted gene (except for TaGASR7) in different arrays of epegRNAs induced by CMPE-ePPEplus. Values were calculated from the data presented in d. Frequencies (mean ± s.e.m.) were calculated from three independent experiments (n = 3) in b, d, and e