Spatial genomics of AAVs reveals mechanism of transcriptional crosstalk that enables targeted delivery of large genetic cargo.

Integrating cell type-specific regulatory elements (e.g. enhancers) with recombinant adeno-associated viruses (AAVs) can provide broad and efficient genetic access to specific cell types. However, the packaging capacity of AAVs restricts the size of both the enhancers and the cargo that can be delivered. Transcriptional crosstalk offers a novel paradigm for cell type-specific expression of large cargo, by separating distally-acting regulatory elements into a second AAV genome. Here, we identify and profile transcriptional crosstalk in AAV genomes carrying 11 different enhancers active in mouse brain. To understand transcriptional crosstalk, we develop spatial genomics methods to identify and localize AAV genomes and their concatemeric forms in cultured cells and in tissue. Using these methods, we construct detailed views of the dynamics of AAV transduction and demonstrate that transcriptional crosstalk is dependent upon concatemer formation. Finally, we leverage transcriptional crosstalk to drive expression of a large Cas9 cargo in a cell type-specific manner with systemically-administered engineered AAVs and demonstrate AAV-delivered, minimally-invasive, cell type-specific gene editing in wildtype animals that recapitulates known disease phenotypes. Highlights:Transcriptional crosstalk between enhancers and promoters delivered in trans by AAVs is a generalized phenomenon. Spatial genomics techniques, AAV-Zombie and SpECTr, reveal that AAV genome concatemerization facilitates transcriptional crosstalk.Transcriptional crosstalk can be leveraged for minimally-invasive, targeted AAV delivery of large cargo, including machinery for CRISPR-based gene editing and manipulation.Transcriptional crosstalk enables cell-type specific gene disruption in wildtype animals, recapitulating behavioural phenotypes of genetic knockouts.