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.
Abstract
Virus-like particles (VLPs) enable transient, non-integrating delivery of CRISPR-Cas9 ribonucleoprotein cargo. Although VLPs have been reported for efficient DNA editing via base editors RNP delivery, the diversity of base editors tested as VLPs remains limited. We generated and benchmarked a panel of 12 base editors on the v5 eVLP backbone, targeting three genomic loci (HEK3, B2M, PDCD1) across five VLP dosages in LentiX-293T cells. Editing efficiency was generally dosage-dependent across all editors and varied by editor class and identity; PAM-flexible variants had lower editing efficiency than NGG-restricted counterparts, and the dual-function SPACE base editors showed reduced efficiency. We further characterized position-specific editing efficiencies and outcomes of the base editor VLP collection, revealing that a wide variety of mutation types are possible with the base editors in this collection.
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.
Jethro Langley, Lou Baudrier, Jada Curry et al.· Cell Genomics· 1 citation
ABSTRACT Despite the promise of DNA base editors for diverse genome editing applications, their utility remains constrained by off‐target effects, which are exacerbated by short spacers in miniature systems and the large size of Cas9‐derived editors, which impedes adeno‐associated virus (AAV) delivery. Here, guided by structural insights into a compact Cas9d nuclease from Deltaproteobacteria, we developed an efficient Cas9d system (Cas9dUltra) through gRNA and protein engineering, and further developed its base editors (9dBEs). Cas9dUltra and 9dBEs enabled efficient and precise genome editing in human cells. Notably, 9dCBE induced premature termination codons in 89% of mouse pups by microinjection, facilitating robust disease modeling. Furthermore, a single AAV delivering 9dCBE achieved efficient Pcsk9 editing and a concomitant reduction in serum LDL‐C levels in mice. Collectively, this study establishes a series of compact, potent genome editing tools poised to advance biological and biomedical translational research.
Qingquan Xiao, Zhijin Tian, Luqi Weng et al.· Advancement of science· 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
The types, principles and characteristics of gene editing systems are introduced in order to understand their requirements for delivery tools and to provide new insights to facilitate appropriate delivery systems or improve the efficacy of existing systems.
Fanyu Xue, Zixuan Xin, Gaojie Wang et al.· Artificial Cells Nanomedicin...· 0 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 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