Background

Since the advent of CRISPR gene scissors, crop genome editing has become a core driver of plant breeding. Prime Editing (PE), which allows for the substitution or insertion of target sequences without DNA double-strand breaks, is gaining attention as a precise tool that reduces the risk of deletions. In monocot grains such as rice and maize, editing efficiencies exceeding 20% have been reported, rapidly advancing toward practical application.

Conversely, research on dicot plants, including soybean, tomato, and rapeseed, has hit a wall. This is due to extreme variability across target sites in dicots compared to monocots, with average editing efficiencies remaining around 1%. The vulnerability of prime editing guide RNA (pegRNA) being easily degraded within plant cells has been repeatedly highlighted. The phenomenon where Reverse Transcriptase (RT) activity drops sharply in dicot plant cell environments also posed a hurdle. The reality was that applying PE to field crop improvement was difficult because intracellular DNA repair mechanisms blocked the introduction of flap sequences.

Key Findings

A research team led by Caixia Gao of the Chinese Academy of Sciences made a breakthrough on this challenge using the 'SmartPE' system, which integrates complex engineering. This achievement was published in the latest issue of the international journal 'Nature Plants'. The team devised a solution to neutralize the interference of the host cell's repair system by precisely designing intentional mismatches within the Reverse-Transcription Template (RTT). They integrated a dual pegRNA system that doubles cleavage efficiency, plant-specific cis-regulatory elements, and an improved reverse transcriptase (RT) with enhanced activity into a single platform.

SmartPE demonstrated overwhelming numerical improvements compared to standard PE in dicot protoplast experiments. In experiments with the model plant Arabidopsis thaliana Col-0, editing efficiency increased by 6.3 to 23.0 times compared to existing methods. In tomato (Solanum lycopersicum) protoplasts, efficiency increased from 8.2-fold to up to 38.2-fold compared to existing systems. In soybean (Glycine max) and rapeseed (Brassica napus), improvements exceeding 10-fold were confirmed at most target sites.

Performance was also confirmed at the organizational level. When editing the SlOR2 gene in a tomato hairy root system, standard prime editing resulted in only 1.0% heterozygous variant formation. In contrast, SmartPE induced 7.3% homozygous variant lines and 12.5% heterozygous lines. This demonstrated the possibility of directly obtaining desired pure-line variants without complex generational advancement.

Practicality was also demonstrated in the stable transformation stage. The research team successfully inserted FLAG tag sequences into the Arabidopsis genome without defects. In tomato plants, enhancer sequences and recombination marker sites were inserted with up to 32.3% efficiency. This lays the groundwork for precisely tuning endogenous gene expression levels without inserting foreign transgenes.

Significance and Outlook

This discovery holds exceptional value as it expands precision editing technology, which was previously focused on monocot grains, to vegetables, legumes, and oilseed crops. It identifies that beyond simple single-base substitutions, regulatory elements or functional tag sequences can be accurately inserted at desired locations. Consequently, research into complex trait improvement, such as introducing pathogen resistance factors and constructing drought-tolerant traits, is expected to gain significant momentum.

Challenges remain to be addressed before these can lead to the development of varieties for dissemination to farms. It remains to be further confirmed across various varieties whether the editing capabilities verified in protoplasts and hairy roots are maintained during the full plant regeneration process. Additionally, whole-genome analysis regarding the stable inheritance of edited traits through generations and potential off-target mutations must be conducted in parallel.