Selector AAV-CRISPR vectors facilitate on-target precise genome editing and purge off-target chromosomal insertions

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 (donor) 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. Moreover, AAV donor constructs can equally yield imprecise on-target edits resulting from end-joining recombination pathways. Here, we demonstrate that endowing AAV vectors with marker-free selectable sequences permits enriching for cells precisely co-edited at endogenous ATP1A1 and target alleles. These selector AAV donors install ATP1A1 polymorphisms conferring resistance to the small-molecule ouabain yielding high frequencies of on-target and precisely edited cell populations. Crucially, we further report that selector AAV donors achieve a thorough removal of heterogeneous off-target DNA species resulting from conventional AAV-based genome editing procedures.