Journal Article

Selector adeno-associated viral vectors facilitate on-target precise genome editing and purge off-target chromosomal insertions

Hoeben, Rob C., Wang, Xiaoling, Liu, Jin, Li, Zhen, Janssen, Josephine M., Gonçalves, Manuel A.F.V.

2026

Overview

Selector adeno-associated viral (AAV) vectors are introduced for retrieving genetically engineered cells, requiring neither heterologous marker chromosomal insertions nor surrogate reporter gene expression systems.Ouabain is a plant-derived cardiac glycoside that inhibits the sodium/potassium (Na+/K+)-ATPase pump. Selector AAV vectors coupled with ouabain treatment enrich for precisely targeted gene knock-ins.Selector AAV vectors coupled with ouabain treatment foster the elimination of off-target exogenous donor DNA insertions from genetically engineered cell populations.In-linkage selector AAV vectors coupled with ouabain treatment yield engineered cell populations with high purity levels of precisely edited cells in a vector dose-independent manner.In trans selector AAV vectors coupled with ouabain treatment permit single-step multiplexing gene knock-ins in hard-to-transfect cells, facilitating complex cell engineering efforts in said cells. 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 highly recombinogenic nature of AAV genomes also facilitates their nonhomologous 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 exogenous marker-free selectable sequences permits enrichment for cells precisely coedited at endogenous target and ATP1A1 alleles. These selector AAV vectors 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 vectors achieve a thorough removal of heterogeneous off-target DNA species resulting from conventional AAV-based genome editing procedures. [Display omitted] The combined effects of enrichment for cells with precisely targeted gene knock-ins with the purging of cells with off-target donor DNA insertions support the adoption of ouabain-based selector adeno-associated viral (AAV) vector systems in basic research and biotechnological settings. In particular, selector AAV vector systems with in cis, in trans, and in-linkage designs form an enabling combinatorial delivery-selection platform that is most readily applicable to in vitro or ex vivo modification of mammalian cells, especially those that are refractory to chemical and physical donor transfections. Indeed, the high cytotoxicity of ouabain is expected to narrow its operational doses in the context of in vivo settings, rendering its application to these settings difficult. For applications that exclusively involve selection in cell culture systems, including transplantation of ex vivo–engineered cell products, the selector AAV–CRISPR platform is at Technology Readiness Level 4 in that it has been effectively used under controlled laboratory conditions for targeted safe harbor gene knock-ins, endogenous gene tagging, and multiplexed gene knock-ins. For applications that include in vivo selection of engineered cells, further developments are required, e.g., identification of compounds displaying wider effective dose windows than that of ouabain. In addition to the limitations associated with the use of ouabain in vivo, direct administration of selector AAV–CRISPR vectors in vivo is hindered by the lack of HR factors in postmitotic cells and tissues. Finally, in common with ‘classic’ AAV-based gene supplementation therapies, application of selector AAV vectors in therapeutic contexts will profit from improving good manufacturing practice (GMP)-grade upscaling and downstream processing of AAV vector batches to maximize cost-effectiveness and product quality, respectively. Cost-effectiveness will improve by establishing more robust transfection-independent AAV production methods, while product quality will increase by implementing downstream processes yielding thorough removal of empty and partially filled AAV particles from the final drug substance. This study establishes selector adeno-associated viral (AAV) vectors for the targeted and precise installation of chromosomal gene-sized edits. The incorporation of a marker-free co-selection system based on a cheap small molecule permits selector AAV vectors to, next to fostering targeted DNA editing, purge off-target chromosomal insertions, improving the quality of engineered cell products.

• Publisher: Elsevier Ltd
• Subject: adeno-associated viral vectors / CRISPR / donor DNA insertions / homology-directed repair / off-target effects / precision gene editing