Efficient and versatile multiplex prime editing in hexaploid wheat

Prime editing is limited by low efficiency in plants. Here, we develop an upgraded engineered plant prime editor in hexaploid wheat, ePPEplus, by introducing a V223A substitution into reverse transcriptase in the ePPEmax* architecture. ePPEplus enhances the efficiency by an average 33.0-fold and 6.4-fold compared to the original PPE and ePPE, respectively. Importantly, a robust multiplex prime editing platform is established for simultaneous editing of four to ten genes in protoplasts and up to eight genes in regenerated wheat plants at frequencies up to 74.5%, thus expanding the applicability of prime editors for stacking of multiple agronomic traits.

Automated summary

Researchers developed an upgraded engineered plant prime editor, ePPEplus, by introducing a V223A substitution into reverse transcriptase in hexaploid wheat, which enhances editing efficiency by an average of 33.0-fold compared to the original PPE and 6.4-fold compared to ePPE. Importantly, they established a robust multiplex prime editing platform for simultaneous editing of multiple genes, achieving editing frequencies up to 74.5% in regenerated wheat plants, thus expanding the applicability of prime editors for stacking agronomic traits.