SmartPE Boosts Prime Editing in Dicot Crops

Engineered templates tackle the dicot efficiency bottleneck

Prime editing allows researchers to install base substitutions, small insertions and deletions without creating double-strand DNA breaks or requiring donor templates. The invention minimizes unintended insertions and byproducts, yet its performance in dicotyledonous crops has remained stubbornly low and erratic across different genomic loci.

To raise expression levels of the editing machinery, a team led by Professor Gao Caixia at the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences first tested regulatory sequences placed between the promoter and the Cas9 sequence. According to Phys.org, inserting a cis-regulatory sequence known as the START element increased transcript levels 2.1-fold and lifted editing rates across four Arabidopsis test sites by roughly 1.8-fold.

A paired prime editing guide RNA design was tested next, but paired guide RNAs alone failed to boost efficiency. The researchers reasoned that tight complementary pairing between the reverse transcriptase template and genomic DNA was hindering enzyme progression, while cellular mismatch repair pathways were suppressing the edits. The team responded by introducing intentional base mismatches into the reverse transcriptase templates of both guide RNAs, a technical innovation that lifted average editing activity at four target sites more than sixfold to about 10%.

Template architecture governs reverse transcription

The research team systematically mapped how the spacing and position of introduced mismatches altered editing outcomes. Tests revealed that continuous mismatches or those placed 3 bp apart delivered the highest performance, reaching efficiencies of up to 20%, as reported by Phys.org. When the distance between mismatches expanded to 6 base pairs or more, editing rates plummeted.

Position also dictated the outcome. Placing mismatched bases at both edges of the editing window improved reverse transcription initiation, whereas placing them directly in the middle produced no benefit. Tests in human cell lines revealed that inhibiting the mismatch repair pathway did not alter SmartPE outcomes in the same way it changed standard single-guide editing, indicating that the dual-guide system relies on alternative cellular repair pathways.

Targeted crop modifications and insertion limits

The researchers validated the tool in crop protoplasts. Six out of nine tomato targets exceeded 10% editing efficiency, nine out of 11 soybean targets cleared 5%, and five rapeseed targets reached or passed 10%, loci where conventional prime editors were almost inactive. By incorporating synonymous codon changes to produce the required template mismatches, the team installed single amino-acid changes in soybean and tomato targets at efficiencies 1.6 to 11.1 times higher than standard methods.

In stable transformation experiments on tomato hairy roots at the SlOR2 locus, SmartPE produced 7.3% homozygous and 12.5% heterozygous edits, whereas conventional prime editing yielded only chimeras and 1.0% heterozygous edits. In Arabidopsis and tomato, the method successfully inserted protein tags, enhancers and recombination sites at rates up to 32%. In soybeans, however, stable transformations yielded only chimeras, indicating that further technology transfer, intellectual property development, and protocol refinement remain necessary before the tool can reliably deliver stable edits in all legume crops.

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