Optimized prime editing for precise gene modification conferring herbicide resistance in sorghum

Dear editor, Maximizing the yield of sorghum (Sorghum bicolor) is challenging due to competition from weeds, particularly wild grasses. Acetolactate synthase (ALS) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) are targets of broad-spectrum herbicides and precise orthologous residue mutations at specific positions each can confer herbicide resistance (Jin et al. 2022). Prime editing (PE) uses CRISPR/Cas9 nickase, enabling precise point mutations without double-strand breaks or DNA donor templates (Anzalone et al. 2019). However, PE application in plants is limited by low editing efficiency. Introducing the R221K and N394K mutations into the nCas9 H840A improved PE efficiency in rice (Oryza sativa), maize (Zea mays), and wheat (Triticum aestivum) (Jiang et al. 2022; Ni et al. 2023; Qiao et al. 2023). Deleting the RNase H domain of the reverse transcriptase Moloney-murine leukemia virus (M-MLVΔRNase H) and/or using its V223A mutant improved editing efficiency in wheat (Ni et al. 2023). In addition, adding a 5'–3' exonuclease directly to the prime editor increased editing efficiency in HEK293T cells (Fig. 1a) (Truong et al. 2024). Similarly, the 5'–3' exonucleases T5E from bacteriophage and UL12 from herpes simplex virus increased knockout and homology-directed repair efficiency in rice and Nicotiana benthamiana (Schreiber et al. 2024; Wang et al. 2025). The stability of prime editing guide RNAs (pegRNAs) also affects editing efficiency, adding evopreQ1 motif engineered pegRNA (epegRNA) stability in rice, wheat, and maize (Xu et al. 2022; Ni et al. 2023).