Fig. 1

Efficiency of prime editing in maize. a Sequences and their relationships of the two ALS genes, the pegRNA targets, the pegRNA RT templates, the pegRNA primer-binding sites (PBS), and the sgRNA targets for nicking the non-edited strands used for the generation of 4 maize prime-editing vectors. The PAMs, nicking sites, RT template and PBS lengths and introduced mutations are indicated. b T-DNA structures of the 4 PE vectors. The pZ1WS and pZ1WS-Csy4 vectors were designed for editing two targets. Each double-PE vector included two expression cassettes for each of the two pegRNAs or sgRNAs. Two strategies were used to express peg1&2: one was based on the Csy4 RNA processing system, and the other was based on tRNA and HDV ribozyme RNA-processing systems integrated with two drivers, polymerase II (35S enhancer-CmYLCV) and III (shortened U6-26) promoters. The structures of the pegRNA and sgRNA expression cassettes in pegR1-sgR1 and pegR2-sgR2 are the same as those in pZ1PE3/3b. The expression of two sgRNAs in sgR1-sgR2 is driven by OsU3p and TaU3p. c Prime-editing efficiencies based on direct sequencing of the PCR products. The values in the parentheses are based on NGS results. The transgenic lines transformed with pZ1WS-Csy4 were not obtained. d Sequencing chromatograms of the PCR fragments from prime-edited plants transformed with pZ1WS. Double peaks represent heterozygous or chimeric mutations, and an asterisk indicates a mutation induced by PE. The first mutation in #15 is the unwanted mutation introduced by the pegRNA scaffold