Aug 2026· Bioengineering & Translational Medicine· 0 citations· 54 references
Medicine
TL;DR
A PepFect14 analog is employed 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).
Abstract
Abstract 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.
A novel genome-wide CRISPR screening strategy that will facilitate the systematic engineering of novel nonviral genome editing delivery methods, where the identified novel gene hits can be further used to increase editing efficiency for other therapeutically relevant cell types.
Shivani Saxena, Meha Kabra, Amr A. Abdeen et al.· bioRxiv· 2 citations
To systematically map cellular factors constraining nonviral genome editing, influencing uptake and intracellular trafficking, we develop a genome-wide CRISPR screening platform linking perturbation of 19,114 genes to editing outcomes in human cells. We identify six negative regulators of delivery whose depletion increases editing efficiency by up to six-fold across diverse payloads, loci, and cell types. We test the top two factors, GJB2 and BET1L, in two distinct human models: correction of a pathogenic adenine base mutation in KCNJ13 and introduction of a cytosine base mutation in the GABAA receptor gene. Depletion of either improves base-editing outcomes by 6-fold, potentially through effects on delivery. In a patient-derived model of retinal channelopathy, knockdown of either gene improves lipid nanoparticle base editing efficiency by over 3.5-fold. This enables functional restoration of Kir7.1 ion channels in a subset of edited cells, highlighting cellular barriers as actionable targets to enhance the potency of genetic therapies. Low editing efficiency of nonviral delivery in post mitotic tissues presents a challenge to the field of gene therapy. Here, authors dissect the genetic regulators of nonviral delivery in post mitotic retinal epithelial cells describe strategies for improved base editor delivery and editing.
Shivani Saxena, Meha Kabra, Amr A. Abdeen et al.· Nature Communications· 0 citations
A virus-like particle (VLP)-based toolkit that delivers diverse CRISPR editing modalities to human monocytes, macrophages and dendritic cells with high efficiency while preserving viability and innate immune responsiveness is presented.
Hyuncheol Jung, Pascal Devant, Carter Ching et al.· Nature Biotechnology· 0 citations
REPLACE is established as a versatile platform for homology-independent sequence replacement and as a complementary approach for genetic correction in settings where homology-directed repair (HDR), base editing (BE) or prime editing (PE) are inefficient or not applicable.
Tu Dang, Alexandra Roman, Anja Zimmer et al.· bioRxiv· 0 citations
An efficient Cas9d system (Cas9dUltra) is developed through gRNA and protein engineering, and its base editors (9dBEs) further developed through gRNA and protein engineering, enabling efficient and precise genome editing in human cells.
Qingquan Xiao, Zhijin Tian, Luqi Weng et al.· Advancement of science· 0 citations
