This work expands the versatility of eVLPs beyond their current in vivo therapeutic applications, demonstrating their promise for high-throughput functional genomics.
Abstract
Summary Engineered virus-like particles (eVLPs) enable transgene-free ribonucleoprotein delivery for genome editing, yet optimized strategies for high-throughput applications remain unexplored. Prime editing enables precise genomic modifications but suffers from limited efficiency. Here, we present PRIME-VLP (Progressive Repeated Infections for Maximized Editing via Virus-Like Particles), which exploits eVLP-mediated editing kinetics through multiple sequential sub-saturating transductions at optimal intervals. PRIME-VLP achieves 1.5- to 2.9-fold improvements in prime editing efficiency across diverse genomic targets and cell types without increasing off-target editing, compromising cellular viability or causing transcriptional perturbations. By decoupling pegRNA and editor delivery through pegRNA-free eVLPs, PRIME-VLP enables pooled prime editing screens and circumvents transgene silencing limitations. Using a 6,000-pegRNA library targeting TP53, PRIME-VLP achieved 2.8-fold higher editing and improved reproducibility compared to conventional lentiviral delivery, identifying TP53 loss-of-function variants conferring Nutlin-3 resistance. This work expands the versatility of eVLPs beyond their current in vivo therapeutic applications, demonstrating their promise for high-throughput functional genomics.
It is revealed that a wide variety of mutation types are possible with the base editors in this VLP collection, revealing that a wide variety of mutation types are possible with the base editors in this collection.
Asfar Lathif Salaudeen, Trevor Shyiak, Carl G. de Boer· bioRxiv· 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
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).
A. du Rand, Courtney Masterson, Daniel J Verdon et al.· Bioengineering & Translation...· 0 citations
Efficient delivery of CRISPR components remains a major determinant of genome editing outcomes. In this study, we compared conventional lipofection with magnetic nanoparticle-assisted gene delivery (magnetofection) for CRISPR-mediated genome editing efficiency using SpCas9 and AsCas12a systems. Based on the average values obtained from multiple independent targets, lipofection resulted in relatively low indel efficiencies, with mean values of average 8.1%–12.47%. In contrast, magnetofection markedly enhanced genome editing outcomes, yielding average indel efficiencies of average 42.29%–45.04%, representing a substantial increase (3.39- and 5.56-fold, respectively) compared with lipofection. This enhancement was consistently observed across both SpCas9-and AsCas12a-mediated editing, indicating that the improved efficiency conferred by magnetic nanoparticle delivery is independent of the nuclease platform. Furthermore, the increased performance of magnetofection was reproducible across multiple genomic loci and cell lines and was also effective under RNP-based delivery conditions, demonstrating its robustness and reliability. In addition to indel-based genome disruption, magnetofection also significantly improved prime editing efficiency (13.95% on average) compared to lipofection (3.81% on average). Overall, our results demonstrate that magnetic nanoparticle-mediated delivery enables highly efficient and reproducible CRISPR genome editing, substantially outperforming conventional lipofection for both indel formation and prime editing. Magnetofection therefore represents a powerful and broadly applicable delivery strategy for next-generation genome editing applications.
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 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