Prime editing enables precise genome modification of a Populus hybrid

Abstract CRISPR/Cas-based genome editing has been extensively employed in the breeding and genetic improvement of trees, yet precise editing remains challenging in these species. Prime editing (PE), a revolutionary technology for precise editing, allows for arbitrary base substitutions and the insertion/deletion of small fragments. In this study, we focused on the model tree poplar 84K ( Populus alba × P. glandulosa ). We used the 2 × 35S promoter to express a fusion protein of spCas9 nickase (nCas9) and engineered Moloney murine leukemia virus (MMLV), and the Arabidopsis thaliana AtU6 promoter to express an engineered PE guide RNA (epegRNA) and Nick gRNA, pioneering the establishment of the Prime Editor 3 (PE3) system in dicot poplar. Single-base substitutions, multiple-base substitutions, and small-fragment insertions/deletions were edited into three endogenous target genes. The desired edits were identified in hygromycin-resistant (transformed) calli at seven out of nine target sites, with an average editing efficiency ranging from 0.1 to 3.6%. Furthermore, stable T 0 plants contained the desired edits at four out of nine targets, with editing efficiencies ranging from 3.6 to 22.2%. Establishment of the PE3 system provides a powerful tool for the precise modification of the poplar genome.