SmartPE enables efficient prime editing in dicot crops

Prime editing (PE) in dicot plants is hindered by low efficiency and locus-dependent variability, limiting its broad application¹. Here we present SmartPE, an optimized PE system that combines a dual-pegRNA strategy with designed mismatched bases in the reverse-transcription templates (RTTs), together with cis-regulatory elements and engineered reverse transcriptases. The editing efficiencies achieved with SmartPE in protoplasts of key dicot crops were substantially higher than those of canonical PE systems: 6.3-23.0-fold in Arabidopsis thaliana (Col-0), 8.2-38.2-fold in tomato (Solanum lycopersicum), with over 10-fold improvements at the majority of target sites in soybean (Glycine max) and oilseed rape (Brassica napus). In tomato hairy roots, SmartPE generated homozygous (7.3%) and heterozygous (12.5%) edit lines at the SlOR2 locus, whereas standard PE produced only heterozygous edits (1.0%) and several chimeras. In stable transformation experiments, SmartPE enabled seamless insertion of a FLAG tag in Arabidopsis and insertion of enhancers and recombination sites in tomato, with editing efficiencies of up to 32.3%. Together, these results show that SmartPE provides an efficient PE framework for dicot species.