Genome editing based on engineered CRISPR systems is advancing rapidly, with the field increasingly moving toward approaches that avoid the induction of mutagenic double-stranded DNA breaks (e.g., RNA-programmable base editing, prime editing, and donor DNA transposition). These nuclease-free strategies often rely on large or multi-component molecular assemblies that can include gene-sized donor DNA substrates. There is, nonetheless, a paucity of vehicles capable of delivering such large and complex genome-editing components effectively and, ideally, in defined stoichiometric ratios. High-capacity adenoviral vector particles (AdVPs) offer an attractive set of features to address these challenges, including robust cell transduction levels regardless of mitotic status, exceptional payload capacity (up to ~36 kb), strict chromosomal nonintegrating character, and the complete absence of viral coding sequences. Hence, AdVPs can serve as biological nanoparticles suitable for the evaluation and application of next-generation CRISPR technologies in physiologically relevant cellular contexts, regardless of the size and number of the attendant tools. Here, after summarizing the key characteristics of earlier- and latest-generation adenoviral vector platforms, we describe protocols for producing AdVPs, including vectors that deliver multiplexing, prime-editing, and orthogonal nuclease constructs. Finally, we highlight important considerations for designing AdVP production reagents and validate a storage buffer that preserves AdVP functionality after repeated freeze-thaw cycles.
Xiaoling Wang, Jin Liu, Josephine M. Janssen et al.· Methods in molecular biology· 0 citations
The Training School on the Impact of GE Delivery Tools on Target Cells was designed to provide an in-depth overview of delivery strategies for genome editing (GE) tools, a cornerstone for gene therapy and cellular engineering. The ability to efficiently and safely introduce genome editing reagents into diverse target cells remains one of the most critical challenges in advancing translational applications of CRISPR, base editing, and related technologies. This three-day program combined theoretical lectures from international experts with complementary laboratory sessions. The lectures covered delivery vectors and nanoparticles, genome editing platforms, and safety and immunological aspects, while the hands-on sessions focused on evaluating delivery efficiency and editing outcomes using different approaches. Participants had the opportunity to explore both conceptual frameworks and experimental techniques — from lipid nanoparticles and viral vectors to genome editing assessment in primary T cells. The interactive structure of the Training School fostered scientific exchange, critical thinking, and practical skill development.
Carla Fuster‐García, Manuel Rhiel, Duško Lainšček et al.· Zenodo (CERN European Organi...· 0 citations
The Training School on the Impact of GE Delivery Tools on Target Cells was designed to provide an in-depth overview of delivery strategies for genome editing (GE) tools, a cornerstone for gene therapy and cellular engineering. The ability to efficiently and safely introduce genome editing reagents into diverse target cells remains one of the most critical challenges in advancing translational applications of CRISPR, base editing, and related technologies. This three-day program combined theoretical lectures from international experts with complementary laboratory sessions. The lectures covered delivery vectors and nanoparticles, genome editing platforms, and safety and immunological aspects, while the hands-on sessions focused on evaluating delivery efficiency and editing outcomes using different approaches. Participants had the opportunity to explore both conceptual frameworks and experimental techniques — from lipid nanoparticles and viral vectors to genome editing assessment in primary T cells. The interactive structure of the Training School fostered scientific exchange, critical thinking, and practical skill development.
Carla Fuster‐García, Manuel Rhiel, Duško Lainšček et al.· Zenodo (CERN European Organi...· 0 citations
We use cookies to run the site and, with your consent, for analytics and to show ads.
See our Cookie Policy.
