Chapter 5 —— 98 —— Abstract Adeno-associated viral (AAV) vectors are commonly used for genome editing owing to the proclivity with which their single-stranded genomes serve as homologous recombination (HR) substrates during programmable nuclease-assisted gene targeting. However, the high recombinogenic nature of recombinant AAV genomes also facilitates their non-homologous end joining at off-target chromosomal breaks (“capture”) created by said nucleases, mutagens, or DNA metabolic processes. The collateral build-up of off-target and random insertions occurs in an AAV dose-dependent manner and greatly diminishes the overall genome-editing accuracy. Moreover, AAV donor constructs can equally yield imprecise on-target edits resulting from non-homologous recombination pathways. Here, we demonstrate that endowing AAV donors with marker-free selectable sequences permits enriching for cells precisely co-edited at target and endogenous ATP1A1 alleles. These selector AAV donors instal ATP1A1 polymorphisms conferring resistance to the small-molecule ouabain and, in the process, yield high frequencies of on-target and precisely edited cell populations independently of the initially applied vector dose (up to 99.4%). Crucially, we further report that next to marker-free enrichment for precisely edited cell populations, selector AAV donors achieve a thorough removal of cells with off-target DNA insertions heightening, therefore, the ultimate precision of AAV-based genome editing. Introduction Genome editing technologies are emerging at a fast pace with their application in scientific and biotechnological realms continuing to expand (Pacesa et al. 2024). Insertion of exogenous (donor) DNA at predefined chromosomal positions (gene targeting or knock-in) subjected to doublestrand DNA breaks (DSBs) made by clustered regularly interspaced short palindromic repeat (CRISPR)-derived nucleases, forms a set of commonly used and highly versatile genome editing principles. This results from the amenability of these gene targeting approaches to large genomic edits (e.g.,
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