Perennial crops supply a large share of the world's food, fiber, and bioenergy, yet they remain stubbornly difficult to improve through modern molecular tools. For many plant species, simply moving a useful edit from the laboratory bench into a whole, fertile plant is the single greatest obstacle to applied research. Efficient plant genetic transformation is a prerequisite for testing gene function, stacking traits, and translating discovery into the field, but most woody and tuber-bearing species regenerate poorly after manipulation. A new strategy tackles this problem head-on: rather than treating regeneration as a separate downstream step, it engineers regenerative capacity into the editing construct itself. The approach, termed CRISPR-Combo, offers a practical route to overcome one of the most persistent bottlenecks in perennial crop biotechnology.
Genome editing begins with the introduction of a nuclease or base editor into living cells, but the edit is only useful if those cells can be coaxed back into a complete organism. In annual model plants such as Arabidopsis or rice, standardized tissue culture protocols make regeneration comparatively routine. Perennial crops tell a different story. Citrus, potato, strawberry, and poplar each carry intrinsic traits—thick cell walls, complex secondary metabolism, and strong developmental inertia—that resist both transformation and the reorganization of cultured tissue into shoots and roots. Even when edited cells are produced, the majority fail to form callus or shoots, and the few that do often require months of hormone treatments under tightly controlled conditions. This recalcitrance raises the time, cost, and failure rate of editing experiments, putting them out of reach for many laboratories.
CRISPR-Combo is built on a straightforward idea: instead of delivering one genetic payload, deliver two that work together in the same cell. The system uses a single construct to perform conventional targeted editing at a gene of interest while simultaneously driving the transcriptional activation of morphogenic genes—the developmental regulators that push plant cells toward regeneration. Unlike conventional CRISPR/Cas9 plant genetic modification, which typically cuts or rewrites one locus and stops there, CRISPR-Combo couples that edit to a built-in regeneration signal. This is achieved through a transcriptional activation module rather than through simple gene overexpression in plants from a separate promoter cassette, allowing the morphogenic program to be activated precisely where and when editing occurs. As a result, the edited cells are simultaneously primed to regenerate into whole plants. Beyond the immediate practical gain, this design expands the conceptual toolkit of plant genetic engineering by showing that developmental competence can be engineered as a component of the editing event rather than left to chance in tissue culture.
Figure 1. Schematic of the improved poplar transformation and genome-editing pipeline enabled by simultaneous WOX11 and WUS activation under hormone-free conditions. (Ramasamy, et al., 2026)
The activation half of CRISPR-Combo relies on the same guide RNA architecture used for editing, but the activation arm uses a dCas9–SunTag scaffold that recruits a GCN4 scFv–2×TAD activator complex, guided by gRNA2.0 (15-nt spacers), to turn on endogenous morphogenic genes instead of cutting DNA. This form of regulation is closely related to the broader family of CRISPR activation (CRISPRa) tools, except that here activation is fused into the editing workflow so that morphogenic genes are turned on as a byproduct of the editing process. Because regeneration is the rate-limiting step, embedding it in the construct means that selection for edited tissue automatically selects for tissue with elevated regenerative capacity. The strategy also dovetails with established somatic embryogenesis and plant regeneration pipelines, offering a complementary lever for species that already rely on embryogenic culture.
Potato was used as the first testbed for identifying which morphogenic genes actually matter. The researchers screened a panel of seventeen candidate regeneration-promoting genes and found that four stood out for their ability to support the regeneration of genome-edited hairy roots: StWOX11/12, StARF5, and two paralogs of ABI3 designated ABI3-1 and ABI3-2. Hairy root systems are valuable because they allow rapid evaluation of edited genotypes without waiting for full plants, and these four regulators consistently improved the recovery of rooted, edited material. Notably, three of the four—StWOX11/12, StARF5, and StABI3-2—also enhanced shoot regeneration through stable transformation mediated by Agrobacterium. This dual benefit is important: a morphogenic gene that helps only one developmental route has limited utility, whereas regulators that improve both root and shoot recovery streamline the entire editing pipeline from initial transformation to fertile plant.
Citrus presents a particularly severe regeneration challenge because most commercial varieties are highly recalcitrant and prone to somaclonal variation. Applying CRISPR-Combo to citrus, the team activated a set of five morphogenic genes—BBM3, FUS3, IPT1, SERK1, and STM—and observed markedly improved plant regeneration from edited tissue. Each of these genes plays a recognised role in embryogenesis or meristem establishment, and their coordinated activation appears to compensate for the developmental rigidity that normally blocks citrus tissue culture. The result is meaningful because citrus improvement has long depended on slow, genotype-dependent protocols; a construct that reliably raises regeneration rates could make editing accessible across a far wider range of varieties, including those that were previously considered impractical to transform. By replacing genotype-specific, experience-driven protocols with a construct-driven process, this work makes citrus editing accessible to more laboratories.
Wild strawberry offered a different kind of advantage: not necessarily higher regeneration frequency, but a dramatically shorter path to edited plants. When the Baby Boom genes BBM1 and BBM2 were activated at the same time as editing, or when GRF3 and GIF1 were activated together, the time required to obtain genome-edited plants dropped by more than a month. BBM transcription factors are well known for their ability to confer pluripotency on plant cells, while the GRF3–GIF1 pair supports meristem growth and organ formation. Their simultaneous activation with the edit meant that selection for the edit selected for tissue already on a fast track to plant formation. For breeders, saving one month per cycle is significant: the gain compounds across generations in accelerated breeding programs, making iterative editing and trait stacking far more feasible within a realistic project timeline.
Poplar, a model woody perennial and an important bioenergy crop, yielded the most striking result. By simultaneously activating WUS and WOX11, the researchers achieved synergistic promotion of regeneration without any exogenous plant hormones in the culture medium. WUS maintains the stem cell pool in the shoot apical meristem, while WOX11 drives regenerative cell division at wound sites; together they appear to recreate the hormonal and developmental cues that tissue culture normally supplies externally. The outcome was genome-edited shoots produced in under one month, a remarkable acceleration for a tree species. Removing the need for externally supplied hormones also reduces protocol complexity and the risk of undesirable responses to hormone regimes, pointing toward a more reproducible, standardized route to editing other woody perennials that share similar regenerative constraints.
The significance of CRISPR-Combo extends beyond the four species demonstrated. The central insight—that regeneration competence can be engineered into the editing construct—is broadly generalizable, because the morphogenic genes identified here belong to conserved developmental pathways found across the plant kingdom. A laboratory working on grape, apple, coffee, or cassava could in principle adopt the same logic, screening local morphogenic regulators and pairing their activation with the edit of interest. This reframing also changes how we think about failed editing experiments: poor recovery may not reflect a problem with the nuclease or guide RNA at all, but rather an unaddressed regeneration block that a morphogenic activation module could resolve. Conceptually, the work shifts the limiting factor of perennial editing from tissue-culture expertise to construct design. It also complements emerging methods that seek to avoid tissue culture altogether, suggesting a future in which edited plants are recovered through multiple, reinforcing routes.
CRISPR-Combo represents a pragmatic and broadly applicable solution to one of the longest-standing obstacles in plant biotechnology: the difficulty of regenerating edited perennial crops. By fusing targeted genome editing with the simultaneous activation of morphogenic genes, the system converts regeneration from a separate, failure-prone step into an integrated feature of the editing event itself. The evidence across potato, citrus, strawberry, and poplar shows consistent, species-appropriate gains—whether in root and shoot recovery, in accelerated timelines, or in hormone-free regeneration of woody tissue. For researchers and biotech practitioners working with recalcitrant species, this approach lowers both the technical and economic barriers to producing genome-edited plants. As morphogenic gene sets are mapped for additional crops, CRISPR-Combo could become a routine tool for editing perennial crops, accelerating the translation of genetic discovery into improved varieties.
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