Production and Storage of High-Capacity Adenoviral Vectors for the Delivery of Advanced CRISPR Systems.

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.