Development of Alfalfa with Combined Herbicide Resistance via Simultaneous Triple Gene Editing using Prime Editing
Background
Alfalfa (Medicago sativa), known as the queen of forages, is a perennial legume widely cultivated as a protein source in livestock farming. It holds high value as forage due to its balanced amino acid composition and rich vitamin and mineral content. However, in packaged cultivation, it has been vulnerable to various weed invasions, facing the dual challenges of reduced yield and degraded quality. In the early growth stages, failure to compete with weeds for sunlight, moisture, and nutrients often leads to failed stand formation.
The most efficient means of weed control is herbicide application. However, there has been an absolute shortage of alfalfa breeding varieties with resistance to commercial herbicides. To control both gramineous and broadleaf weeds invading single-crop fields, multiple herbicides with different modes of action must be used in combination. Existing alfalfa varieties are highly sensitive to herbicides, suffering severe injury even with slight misuse of herbicides. Even when attempting to introduce target traits, it was difficult to confer complex resistance in a short period through traditional cross-breeding due to the complex genetic structure unique to tetraploid crops. This created an urgent need for precision molecular breeding technology using gene editing.
Key Findings
Researchers designed a Prime Editing (PE) platform that increased expression efficiency within alfalfa cells and succeeded in simultaneously editing three target genes present in the crop genome.
The target genes for editing were the acetolactate synthase genes MsALS1 and MsALS2, and the acetyl-CoA carboxylase gene MsACC1. Based on previous research results on rice homologs, mutations conferring ALS resistance to sulfonylureas and other compounds were designed into MsALS1 and MsALS2. This was intended to provide resistance to nicosulfuron, commonly used in maize and soybean cultivation. Precise point mutations were also planned for the MsACC1 gene to confer resistance to aryloxyphenoxypropionate (APP) class herbicides.
To edit the three genes simultaneously in a single transformant, the researchers introduced the Csy4 nuclease system derived from bacteria for RNA processing. The principle involves expressing a multi-pegRNA transcript linked by Csy4 recognition sequences within the cell, where the Csy4 protein cleaves each RNA into individual units to form multiple complexes. Through this system, the intended base substitutions occurred precisely in the three alfalfa genes. The gene-edited alfalfa lines, in which the mutations were stably established, maintained normal growth even in greenhouse tests involving the combined application of nicosulfuron and the APP-class herbicide haloxyfop-P-methyl.
Significance and Outlook
This achievement, which precisely substituted three genes in a single cell without off-target deletions, is regarded as a breakthrough that has overcome the molecular breeding barrier for tetraploid perennial crops. In crops like alfalfa, which have high chromosome copy numbers and complex genetic variations, correcting with conventional gene editors that induce double-strand breaks (DSBs) often resulted in unexpected deletions or chromosomal rearrangements. This study demonstrated that by combining the PE method, which directly overwrites base sequences, with a multi-target system, it is possible to induce desired genotypes while minimizing genomic damage.
At the farm level, customized control becomes possible by selectively combining graminicide (haloxyfop-P-methyl) and broadleaf/graminaceous dual-purpose herbicides (nicosulfuron). This widens the scope for implementing cross-control systems without crop damage when herbicide-resistant weeds emerge.
However, there are still many challenges to overcome before reaching the packaging demonstration stage. It is necessary to determine whether foreign expression cassettes used in the transformation process persist, and measures to prevent gene flow via pollination must be established. As regulatory standards for gene-edited crops vary by country, securing null-segregant lines and verifying yield and nutritional value will determine the timeline for commercialization.
Alfalfa (Medicago sativa) is the most widely cultivated forage crop worldwide, owing to its high protein content, balanced amino acid profile, and abundance of vitamins and minerals. However, weed infestation severely threatens alfalfa yield and quality. The lack of herbicide‑resistant cultivars, combined with the diversity of weed species, has made herbicide tolerance a key breeding objective. To address this challenge, we developed a highly optimized prime editing system and, for the first time, achieved prime editing in alfalfa by enhancing the expression efficiency of its components. Specifically, we introduced mutations in three target genes MsALS1, MsALS2 and MsACC1 to confer herbicide resistance: mutations in MsALS1 and MsALS2 were designed to confer resistance to ALS‑inhibiting herbicides (based on evidence from their rice homologs), while those in MsACC1 were targeted to confer resistance to aryloxyphenoxypropionate (APP) herbicides. Using the Csy4 nuclease system, we performed simultaneous prime editing of all three genes, ultimately generating alfalfa lines resistant to nicosulfuron and haloxyfop‑P‑methyl. This study represents the first successful multi‑gene prime editing in alfalfa, providing a novel tool and a valuable approach for alfalfa molecular breeding.
This research serves as a practical catalyst for increasing productivity in large-scale alfalfa seed production complexes and commercial forage production farms. In existing alfalfa cultivation fields, the burden of labor and herbicide costs was high due to reliance on pre-plowing or limited selective herbicides for early weed control. With the introduction of the developed multi-resistance lines, herbicide combinations used in rice or maize cultivation can be flexibly applied to alfalfa fields.
It can prevent the decline in crude protein content of harvested forage caused by missing the control window immediately after germination and maximize mechanized harvesting efficiency. The multiplex prime editing protocol provides a technological foundation that can be immediately applied to the molecular breeding of other crops to simultaneously improve complex agricultural traits such as drought resistance, lodging resistance, and reduced lignin content.