Fig. 1

Optimized pegRNA by incorporation of different modifications at its 3′ extension. a Schematic diagram of engineering a prime editor by two aspects: optimizing pegRNA component by adding different modifications at 3′ extension of pegRNA, and engineering prime editor protein component by mutation of reverse transcriptase and optimization of prime editor architecture. NC, viral nucleocapsid protein. M-MLV RT, Moloney-murine leukemia virus reverse transcriptase. PBS, primer binding site. RTT, reverse transcription template. b Schematic representation of canonical pegRNA and six modified pegRNAs including pegRNA-Csy4RS, epegRNA, pegRNA-ENE, pegRNA-dENEs, pegRNA-U-A·U, and pegRNA-Vc2 constructs. c Frequencies of prime editing and byproducts induced by canonical pegRNAs and modified pegRNAs at seven wheat target sites. Frequencies (mean ± s.e.m.) were calculated from three independent experiments (n = 3). d Summary of the fold change in prime editing efficiencies for modified pegRNAs compared to canonical pegRNAs. Values were calculated from the data presented in c. The editing frequencies using canonical pegRNA for each target were normalized to 1, and the frequencies using other pegRNAs for each target were adjusted accordingly. P values were obtained using the two-tailed Student’s t test: *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001