SmartPE boosts prime editing efficiency in dicot crops

Researchers at the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences have developed a gene-editing system named SmartPE that significantly improves the efficiency of prime editing in dicot plants. The study, led by Prof. GAO Caixia, was published in Nature Plants on October 28, 2026. This advancement addresses a major limitation of current prime editing techniques, which often exhibit low and variable efficiency in dicot species such as tomatoes, soybeans, and rapeseed. By introducing specific modifications to the editing machinery, the team has created a more reliable tool for introducing precise genetic changes into these important agricultural crops.

Prime editing is a versatile gene-editing method capable of installing base substitutions and small insertions or deletions without causing double-strand breaks or requiring a donor DNA template. It is generally associated with relatively few off-target events and byproducts. However, its application in dicot plants has been hindered by inconsistent performance, where editing efficiency varies widely among different target sites. The new SmartPE system aims to overcome these barriers by enhancing the fundamental components of the prime editor to ensure more consistent and higher levels of editing activity across various dicot species.

The core innovation of SmartPE involves introducing mismatches into the reverse transcriptase templates, or RTTs, of two prime editing guide RNAs, known as pegRNAs. This strategy is combined with a cis-regulatory element and an engineered reverse transcriptase. The researchers initially tested cis-regulatory elements to increase editor expression, finding that the START element performed best. It raised transcript levels by 2.1-fold and improved editing efficiency at four Arabidopsis targets by approximately 1.8-fold. This initial improvement in expression provided a foundation for further optimizing the editing mechanism itself.

Building on the expression improvements, the team investigated why a dual-pegRNA strategy had previously failed to enhance editing efficiency. They reasoned that complementary pairing between the RTT and genomic DNA might hinder the reverse transcription process, and that mismatch repair mechanisms could suppress prime editing. To test this, they introduced complementary mismatched bases into the RTTs of both pegRNAs. This modification resulted in a substantial increase in average editing activity at the four tested targets, boosting it more than sixfold to reach approximately 10 percent. This modified system was subsequently named SmartPE.

The researchers further optimized the arrangement of these mismatches to maximize efficiency. They found that continuous mismatches or those spaced three base pairs apart yielded the highest editing efficiency, reaching up to 20 percent. In contrast, intervals of six base pairs or more sharply reduced efficiency. This pattern differed from observations in single-pegRNA systems. Additionally, mismatches located at both ends of the editing window improved efficiency, whereas those in the middle did not. These findings suggest that the specific placement of mismatches may influence the initiation of reverse transcription, providing a practical framework for designing future prime editors.

In cell-based assays, the team examined the interaction between SmartPE and mismatch repair pathways. In U2OS and HeLa cells, the MMR-inhibiting SB protein enhanced single-pegRNA editing but did not enhance SmartPE. This indicates that the two strategies rely on different repair pathways, although the underlying molecular mechanisms remain to be further elucidated.

The practical utility of SmartPE was demonstrated in protoplast assays across several dicot crops. In tomato, six of nine targets exceeded 10 percent editing efficiency. In soybean, nine of 11 targets exceeded 5 percent efficiency. In rapeseed, five targets reached or exceeded approximately 10 percent efficiency. Conventional prime editing was nearly inactive at some of these targets, highlighting the significant improvement offered by the new system. Using synonymous substitutions to create RTT mismatches, the team also generated single amino-acid substitutions in soybean and tomato targets at efficiencies 1.6 to 11.1 times higher than standard methods.

Stable transformation experiments further validated the system’s capabilities. In tomato hairy roots, SmartPE produced homozygous edits at 7.3 percent and heterozygous edits at 12.5 percent at the SlOR2 locus, whereas standard prime editing yielded only heterozygous edits at 1.0 percent and chimeras. In Arabidopsis and tomato, the system achieved seamless FLAG-tag, enhancer, and recombination site insertions at up to 32 percent efficiency, including 17 percent co-editing at two targets. However, in soybean, only chimeras have been obtained so far, indicating a need for further investigation. Overall, SmartPE provides a robust framework for applying prime editors in dicot crops, with empirical design principles regarding mismatch density and distribution offering a practical guide for researchers.

Subject of Research: Development of SmartPE, a gene-editing system that enhances prime editing efficiency in dicot plants.

Article Title: Scientists develop new system to boost prime editing in dicot plants

Article References: Scientists develop new system to boost prime editing in dicot plants. Original publication (Bibliographic details unavailable.)

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Keywords: gene editing, prime editing, dicot plants, SmartPE, crop improvement, genetics, tomato, soybean, rapeseed, Nature Plants, Scientists, develop