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