Scientists develop new system to boost prime editing in dicot plants

Scientists from China's Institute of Genetics and Developmental Biology have created a new gene-editing system called SmartPE. This innovation boosts prime editing efficiency in dicot plants like tomatoes, soybeans, and rapeseed, overcoming an earlier limitation with the technique. Prime editing can introduce base substitutions and small insertions or deletions without double-strand breaks or a donor template, resulting in fewer off-target events and byproducts. However, its efficiency is low and varies among target sites in dicot plants.

The researchers enhanced prime editing by inserting cis-regulatory elements between the promoter and Cas9 coding sequence, which increased editor expression by 2.1-fold. They then tested a dual-pegRNA strategy, but found editing efficiency remained unchanged. To address this, they introduced mismatched bases into the reverse transcriptase templates (RTTs) of both pegRNAs. This modification increased average editing activity at four targets by more than sixfold, reaching approximately 10%.

Further experiments revealed that continuous mismatches or those spaced 3 base pairs apart yielded the highest editing efficiency, while intervals of 6 bp or more sharply reduced efficiency. SmartPE's design principles provide a practical framework for applying prime editors in dicot crops. However, stable transformation experiments showed that SmartPE produced homozygous edits in tomato hairy roots at 7.3%, and heterozygous edits at 12.5%, compared to only 1.0% heterozygous edits and chimeras with standard prime editing.

In Arabidopsis and tomato, SmartPE achieved seamless FLAG-tag, enhancer, and recombination site insertions at up to 32% efficiency. In soybean, only chimeras have been obtained, indicating a need for further investigation.