A PepFect14 Analog Improves Non-Viral CRISPR Delivery in Primary Human Cells to Facilitate Genome Editing and Repair.

CRISPR-based designer nucleases can facilitate genome engineering targeting almost any genomic locus. However, safe and efficient methods for delivering gene editors into primary human cells and tissues remain a central challenge. In this study, we employed a PepFect14 (PF14) analog, PF14-K, to deliver high-fidelity Cas9-ribonucleoproteins and non-viral repair templates into primary human skin cells to mediate gene editing and repair targeting genes underlying the group of genetic skin blistering disorders epidermolysis bullosa (EB). Peptide-RNP nanoparticles enabled consistent gene editing of >70% in primary wild type fibroblasts and >50% in primary wild type keratinocytes. In more difficult-to-transfect primary EB skin cells, this strategy facilitated up to 68% exon deletion-mediated reframing targeting COL7A1 and 37% precise homology-directed repair of a prevalent LAMB3 mutation. Compared to electroporation, the gold standard for ex vivo delivery, PF14-K enabled similar total yields of edited cells. Deliverable PF14-K nanoparticles are highly cost-effective, as they can be formed on the benchtop through a simple mix-and-incubate approach, with future potential to deliver base and prime editors.