Agrobacterium is the workhorse of plant transformation, ferrying foreign DNA into plant cells through a process that has been harnessed for decades in laboratories worldwide. Dozens of engineered strains exist, each with distinct genotypes and phenotypes suited to different plant species, yet the molecular details of how the bacterium infects plants and integrates T-DNA into host genomes remain incompletely understood. Screening mutant strains and deliberately constructing engineered variants are considered key routes to unraveling these mechanisms, but the tools for doing so have been cumbersome.

Traditional Agrobacterium gene editing relies on plasmids carrying suicide genes, such as the widely used pK18mobSacB system, and typically demands two rounds of homologous recombination. In the first round, an antibiotic like kanamycin forces a tool plasmid that cannot replicate autonomously in Agrobacterium to integrate into the bacterium’s endogenous Ti or Ri plasmid through homologous recombination. In the second round, the SacB suicide gene enables negative selection against cells still carrying the plasmid, allowing researchers to isolate products of the second recombination event. The problem is that these steps yield large numbers of unmodified bacteria, driving overall modification efficiency down to very low levels.

The new method, described for the first time in detail by Yu Liu, Geng Sun, and colleagues, sidesteps much of this complexity by requiring only a single round of homologous recombination. The trick lies in turning the bacterium’s own biology against itself, so to speak: instead of relying solely on an introduced template, the researchers induce the production of endogenous T-DNA within the Agrobacterium cell and use this single-stranded DNA as the homologous repair template. Because single-stranded DNA is known to be a more effective substrate for homologous recombination than double-stranded DNA, this endogenous supply of T-DNA provides a ready-made molecular scaffold for precise editing.

Getting tool plasmids into Agrobacterium has historically been its own bottleneck, often requiring triparental mating, a three-strain conjugation dance, or expensive electroporation equipment. The Bozhou team showed that a simple freeze-thaw method suffices for introducing their plasmids into competent GV3101 cells, a convenience that could make the protocol accessible to laboratories without specialized transformation apparatus. The researchers constructed a backbone vector called pSG-SacB05, combining pBBR1 origin and replication sequences from pBBR1MCS-2, the SacB gene from pK18mobSacB, a pBR322 origin for easier plasmid preparation, a kanamycin resistance gene, and T-DNA repeat sequences with multiple cloning sites for inserting template sequences.

To demonstrate the method, the team targeted VirD2, a crucial protein in Agrobacterium-mediated genetic transformation that plays a pivotal role in transferring T-DNA from bacterium to plant cell and is essential for integrating T-DNA into the host genome. Using a tool plasmid called pSG-SacB10, they engineered simultaneous modifications at both the N-terminal and C-terminal ends of VirD2, which resides on the Ti plasmid of GV3101. The design inserted a 72-base-pair SpyTag sequence flanked by a 487-base-pair left homology arm derived from VirD1, and deleted 714 base pairs of the VirD2 C-terminus while inserting 152 base pairs of VirF sequence, all flanked by a 574-base-pair right homology arm from VirD3. A 630-base-pair N-terminal segment of VirD2 was preserved and could act as a middle homology arm.

The modification protocol unfolds across eleven carefully defined steps, beginning with vector construction using homologous arms longer than 400 base pairs, followed by freeze-thaw transformation onto plates containing rifampicin, streptomycin, and kanamycin. The critical induction phase involves culturing kanamycin-resistant clones in liquid medium adjusted to pH 5.5 with 20 milligrams per liter kanamycin and 300 micromolar acetosyringone, a phenolic compound that triggers Agrobacterium’s virulence machinery, at 25 degrees Celsius for three days. An optional extended culture step can push modification efficiency higher, and dual PCR detection with specific primer pairs confirms whether edits occurred at both target sites before the bacteria are plated on sucrose-containing medium to eliminate cells still carrying the tool plasmid.

The results revealed a striking dependence on culture conditions. When five independent modification cultures were grown at pH 5.5 with acetosyringone, four produced clear 572-base-pair and 793-base-pair electrophoretic bands indicating edits at both the upstream and downstream regions. Among 22 monoclonal colonies tested from each culture, 13.6 percent carried the downstream modification, 22.7 percent carried the upstream modification, and 4.5 percent carried both simultaneously. After secondary colony purification and SacB residue testing, all 27 sucrose-surviving colonies were SacB negative, and Sanger sequencing of four candidate modified strains confirmed their sequences matched the designed target with perfect fidelity. Kanamycin sensitivity tests further verified that no residual antibiotic resistance from the tool plasmid remained in the successfully edited bacteria.

In contrast, parallel cultures grown at pH 7.0 without acetosyringone told a different story. While 39.1 percent of colonies showed the upstream modification, not a single colony among 110 tested displayed the downstream edit, despite weak 793-base-pair signals in liquid culture. This opposite trend, with upstream modification rising while downstream modification vanished, suggests that small-fragment and long-fragment recombination operate through fundamentally different mechanisms. The researchers propose that the medium-copy-number pSG-SacB10 plasmid provides enough template for short-fragment recombination on its own, but long-fragment modification depends on the abundance of single-stranded endogenous T-DNA, which is only produced under the acidic, acetosyringone-induced conditions that activate Vir gene expression and promote T-DNA processing and stability.

The authors acknowledge that the method’s efficiency, while promising, remains modest, and they point to several contributing factors. High concentrations of streptomycin and kanamycin may suppress Vir gene expression, and more importantly, commonly used Agrobacterium strains including GV3101 carry a function-less recA mutation, removing a key enzyme in DNA repair that would normally boost recombination. The team suggests that introducing a functional recA gene will be crucial for improving efficiency and expanding the host range of the technique in future work. Even so, with nine Sanger-confirmed colonies across multiple experiments all showing 100 percent sequence accuracy, the precision of the method is clear, and given how rapidly bacteria grow, the researchers argue the approach can meet practical demands. They position the protocol as a straightforward, reproducible tool for Agrobacterium genome engineering with potential applications across biotechnology and agricultural sciences, from creating integration-less strains for cleaner plant transformation to building custom variants that illuminate the still-mysterious mechanics of nature’s own genetic engineer.

Subject of Research: A one-step homologous recombination method for precise Agrobacterium genome editing using induced endogenous T-DNA

Article Title: Precise genome modification of agrobacterium based on one-step homologous recombination via endogenous transfer DNA

Article References: Liu, Y., Sun, G., Gao, C., Gao, R., Tang, J., Wang, M., Guo, J., & Guo, R. (2026). Precise genome modification of agrobacterium based on one-step homologous recombination via endogenous transfer DNA. Acta Biochimica Polonica, 73, Article 16599. https://doi.org/10.3389/abp.2026.16599

Image Credits: AI Generated

DOI: 10.3389/abp.2026.16599

Keywords: Agrobacterium, genome editing, T-DNA, homologous recombination, VirD2, SacB, acetosyringone, plant transformation, biotechnology, GV3101, genetic engineering, homology-directed repair

The delivery strategy is disarmingly simple. Preformed high-fidelity Cas9-ribonucleoproteins are mixed with a molar excess of the peptide in a buffer containing the crowding reagent PEG-PVA, which encourages complexation. The cationic peptide electrostatically binds the negatively charged guide RNA within the RNP, forming nanoparticles that cells take up through a route involving class A scavenger receptors. Ornithine residues built into the peptide’s sequence shield it from degradation by serum proteases. Gel shift assays confirmed that even the lowest peptide ratios immobilized the RNPs, and transmission electron microscopy revealed substantially enlarged nanoparticles at a 1:100 molar ratio compared with RNPs alone. Confocal microscopy using red-fluorescent Cas9 showed punctate intracellular signal within one hour, consistent with endosomal entrapment, followed by diffuse cytoplasmic and nuclear fluorescence by 24 hours.

Optimization began with a titration of Cas9-RNP concentrations, from 10 to 17 nanomolar, against peptide molar ratios spanning 1:50 to 1:200. Higher doses drove editing rates up to roughly 75 percent in fibroblasts and 70 percent in keratinocytes but devastated viability, dropping to as low as 9 percent at the most aggressive condition in keratinocytes. The sweet spot emerged at 10 nanomolar Cas9-RNP with a 1:100 peptide ratio, which preserved about 90 percent viability in fibroblasts and 86 percent in keratinocytes, compared with 74 and 66 percent respectively for electroporation. Although electroporation edited a larger fraction of surviving cells, the superior survival of peptide-treated cultures yielded comparable total numbers of edited cells.

Gene expression profiling added a biological argument for gentleness. Using droplet digital PCR 24 hours after editing, the team found that electroporation upregulated CDKN1A, a marker of cell cycle arrest, while suppressing BUB1, a master regulator of mitotic spindle assembly, in fibroblasts. Most strikingly, electroporated fibroblasts showed elevated TGFB1 expression, a hallmark of fibroblast activation and differentiation into myofibroblasts. Because TGF-beta1 is a major driver of fibrosis and squamous cell carcinoma progression in recessive dystrophic epidermolysis bullosa, seeding engineered skin grafts with electrically stressed, myofibroblast-prone cells could undermine clinical outcomes. Peptide delivery perturbed these pathways to a noticeably smaller extent.

A single amino acid proved consequential. Adding one C-terminal lysine to PepFect14 produced PF14-K, which delivered a consistent, roughly 10 percent improvement in editing across three guide RNAs compared with the parent peptide. Structural analysis offered a possible explanation: circular dichroism spectroscopy showed slightly elevated alpha-helical content in PF14-K, and dynamic light scattering revealed larger RNP-peptide particles, around 130 nanometers versus 90 nanometers for the original peptide, with similar polydispersity. The longer, positively charged lysine side chain may strengthen interaction with the RNP’s negatively charged surface. By contrast, a variant in which the N-terminal stearic acid was replaced by oleic acid lost activity across all tested guides, consistent with the idea that the saturated fatty acid’s straight tail promotes tight hydrophobic packing that stabilizes the nanoparticles, while oleic acid’s double-bond kink disrupts it.

Solvent and serum conditions also mattered. Dissolving the peptide in 90 percent ethanol with 10 percent DMSO, rather than water, yielded subtle but reproducible editing gains, echoing prior observations that this solvent combination better solubilizes both the charged and hydrophobic faces of amphipathic peptides and discourages formation of large micellar aggregates. Despite the peptide’s ornithine-stabilized serum resistance, transfection in serum-free medium improved editing by 15 to 20 percent, with two of three guide RNAs reaching statistical significance. Under fully optimized conditions, PF14-K routinely achieved 70 to 75 percent editing in primary fibroblasts and 50 to 60 percent in keratinocytes, at viability exceeding 80 to 90 percent, versus roughly 70 percent for electroporated cultures.

The therapeutic applications were tested directly in patient-derived cells. For recessive dystrophic epidermolysis bullosa, which stems from COL7A1 mutations, the team used dual Cas9-RNPs to excise mutation-bearing exons and restore the reading frame. In patient keratinocytes carrying a heterozygous single-base deletion within exon 31, PF14-K achieved deletion efficiencies up to 58.4 percent by Nanopore amplicon sequencing, and up to 67.8 percent in patient fibroblasts. Electroporation reached 85 to 90 percent but at the cost of viability averaging just 50.7 percent in patient fibroblasts and 63.3 percent in patient keratinocytes, cells already fragile and slow-growing. Flow cytometry confirmed restoration of COL7 protein in up to 61 percent of peptide-edited keratinocytes. Because 20 to 35 percent editing is considered sufficient for therapeutic benefit in epidermolysis bullosa, the peptide-edited cell populations, being both sufficiently corrected and substantially healthier, could represent a superior graft product.

The most technically demanding result was precise homology-directed repair. Using short single-stranded repair templates co-delivered with the RNPs, the researchers corrected a prevalent nonsense mutation in LAMB3, c.1903C>T, which underlies junctional epidermolysis bullosa. In primary patient keratinocytes, the baseline 3:1:100 molar ratio of template to RNP to peptide achieved 14 percent mutation correction. Raising the peptide ratio to 3:1:150 lifted silent marker incorporation to 48 percent, and adding small-molecule inhibitors of the DNA repair enzymes polymerase theta and DNA-PK pushed precise correction to 37 percent by CRISPResso2 analysis, with electroporation reaching up to 51 percent, or 65 percent with inhibitors, but again with lower viability. Notably, the authors state this is the first demonstration of HDR-mediated repair of a pathogenic mutation, or exon skipping, in primary human cells using a non-viral cell-penetrating peptide strategy; earlier peptide HDR efforts were confined to transfection-permissible cell lines, reporter constructs, or safe-harbor loci.

Caveats remain. The AZD7648 DNA-PK inhibitor used in the dual-inhibition scheme has been linked to large-scale genomic alterations in other contexts, so genome-wide off-target assessment will be essential, even though pairing it with a polymerase theta inhibitor has been shown to mitigate genotoxicity. The gene expression analysis covered only a small panel of markers, and three-dimensional skin models will be needed to test whether PF14-K can deliver editors into intact tissue; prior work showed the parent peptide can deliver miRNA via subcutaneous injection in mice, an encouraging precedent. Still, the mix-and-incubate approach requires nothing more exotic than a commercially synthesized peptide and preformed RNPs, sidestepping the packaging limits and insertional risks of viral vectors, the specialized hardware and cytotoxicity of electroporation, and the microfluidics and mRNA-manufacturing burden of lipid nanoparticles. With base and prime editors increasingly bypassing the need for DNA templates altogether, the researchers argue that PF14-K and its successors could extend non-viral genome editing to a broad range of hard-to-transfect primary cells, and perhaps one day, directly to the skin itself.

Subject of Research: Non-viral CRISPR/Cas9 delivery into primary human skin cells using a PepFect14 cell-penetrating peptide analog for genome editing and repair of epidermolysis bullosa mutations

Article Title: A PepFect14 analog improves non‐viral CRISPR delivery in primary human cells to facilitate genome editing and repair

Article References: du Rand, A., Masterson, C., Verdon, D., Siow, A., Loef, E., Dunbar, R., Kingston, R., Harris, P., & Sheppard, H. (2026). A PepFect14 analog improves non‐viral CRISPR delivery in primary human cells to facilitate genome editing and repair. Bioengineering & Translational Medicine, Article e70172. https://doi.org/10.1002/btm2.70172

Image Credits: AI Generated

DOI: 10.1002/btm2.70172

Keywords: CRISPR, Cas9 ribonucleoproteins, cell-penetrating peptides, PepFect14, PF14-K, epidermolysis bullosa, COL7A1, LAMB3, homology-directed repair, gene therapy, keratinocytes, non-viral delivery