Abstract

Prime editing (PE), as a versatile CRISPR/Cas-based precise genome-editing technology, enabled targeted insertions, deletions, and all 12 types of point mutations without double-strand breaks (DSBs) or DNA donor templates. However, the application of PE in plants has been limited due to relatively low editing efficiency. Here, we report that the systematic optimization of all modules in the prime editing toolkit increased editing efficiency in sorghum protoplasts from ~1% to over 9%. Key innovations include the use of a high-activity nCas9 R221K/N394K/H840A variant fused to M-MLV^{ΔRNase H}, the addition of the 5′–3′ exonuclease T5E, and the use of a composite promoter (35S-CmYLCV-SbU6) to drive the expression of engineered pegRNAs. Stable transformation of the elite sorghum cultivar led to the precise generation of the SbALS^W545L and SbEPSPSD208N point mutations, with editing frequencies of 6.3% and 7.7%, respectively. Plants harboring the SbALS^W545L mutation carry no foreign DNA, exhibit resistance to bispyribac-sodium, and show no significant differences in all tested agronomic traits. Our study expands the application of prime editing to sorghum and provides new herbicide-resistant sorghum germplasm, thereby accelerating sorghum breeding.

Plant physiology.Volume 129,IF=6.9.

DOI:https://doi.org/10.1093/plphys/kiag264