A new approach to a persistent plant-editing problem
Researchers in China have developed a gene-editing system intended to improve prime editing in dicot plants, a broad group that includes tomatoes, soybeans and rapeseed. The system, called SmartPE, was reported by a team led by Gao Caixia at the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences. The study was published in Nature Plants on October 28.
Prime editing is designed to make targeted genetic changes, including base substitutions and small insertions or deletions, without creating double-strand DNA breaks or requiring a donor DNA template. The approach can also produce relatively few off-target events and unwanted byproducts. But in dicot plants, editing efficiency has been low and highly variable from one target site to another, limiting how broadly the technique can be used.
SmartPE combines several modifications aimed at addressing that limitation. The researchers added a cis-regulatory element to increase expression of the editing machinery, used two prime-editing guide RNAs, and altered the reverse-transcriptase template regions of those guide RNAs by introducing mismatches.
Increasing expression before changing the guide design
The researchers first tested cis-regulatory elements placed between the promoter and the Cas9 coding sequence. Their goal was to raise expression of the editor itself. Among the elements they examined, the START element performed best. It increased transcript levels 2.1-fold and raised editing efficiency by about 1.8-fold at four Arabidopsis targets.
That result supplied one component of the eventual system, but it did not solve the efficiency problem by itself. The team then tested a dual-guide strategy using two prime-editing guide RNAs, or pegRNAs. On its own, the dual-pegRNA approach did not improve editing efficiency.
The researchers proposed two possible barriers. Pairing between the reverse-transcriptase template and genomic DNA could interfere with reverse transcription, while mismatch repair could suppress prime editing. They therefore redesigned the reverse-transcriptase templates of both pegRNAs to include complementary mismatched bases.
Mismatches made a substantial difference
With those changes, average editing activity at the four tested targets increased more than sixfold and reached about 10%. The researchers named the resulting platform SmartPE.
The position and spacing of mismatches mattered. Continuous mismatches, or mismatches separated by three base pairs, delivered the highest editing efficiencies in the reported tests, reaching as much as 20%. When the gaps between mismatches were six base pairs or more, editing efficiency fell sharply.
This is an important practical detail because prime editing is not simply a matter of selecting a desired genetic change. The activity of the system depends on how the guide and template are configured at a particular target. SmartPE suggests that deliberate mismatch placement can be used as a design variable rather than treated only as a source of error.
Why dicot performance matters
Dicot plants include major food and oilseed crops. A more effective system for making small, precise edits could give researchers another route for studying gene function and developing crop traits. The reported work specifically frames SmartPE as a way to make precise changes more accessible in plants such as tomato, soybean and rapeseed.
The study does not mean that editing performance will be identical across all genes, varieties or crop species. The source material emphasizes that prime-editing efficiency in dicots has varied widely among target sites. Still, the reported gains indicate that low efficiency is not an immovable constraint: changes to editor expression and guide-template design can materially affect the result.
SmartPE also retains the central appeal of prime editing. Rather than relying on double-strand breaks, it is intended to install narrowly defined edits such as substitutions and small insertions or deletions. For plant researchers, that makes the approach potentially useful where the desired outcome is a specific sequence change rather than a broad disruption of a gene.
A platform result, not a finished product
The significance of the work lies in its engineering strategy. The team combined a regulatory change that raised editor expression with an alteration to the guide-RNA templates that appears to reduce barriers to editing. Neither step alone captures the full result. Together, they produced the SmartPE system and the reported increase in activity.
Future work will determine how consistently the approach performs across additional targets and plant species. For now, the study offers a concrete route for improving prime editing in dicots: raise editor expression, use paired guide RNAs, and carefully tune mismatch placement in the reverse-transcriptase templates.
That combination could help shift prime editing in dicot crops from a technique constrained by uneven efficiency toward a more usable tool for precise plant genetics.
This article is based on reporting by Phys.org. Read the original article.
Originally published on phys.org
