Technical and biological sources of noise confound multiplexed enhancer AAV screening

Cis-acting regulatory enhancer elements are valuable tools for gaining cell type-specific genetic access. Leveraging large chromatin accessibility atlases, putative enhancer sequences can be identified and deployed in adeno-associated virus (AAV) delivery platforms. However, a significant bottleneck in enhancer AAV discovery is charting their detailed expression patterns in vivo, a process that currently requires gold-standard one-by-one testing. Here we present a barcoded multiplex strategy for screening enhancer AAVs at cell type resolution using single cell RNA sequencing and taxonomy mapping. We executed a proof-of-concept study using a small pool of validated enhancer AAVs expressing in a variety of neuronal and non-neuronal cell types across the mouse brain. Unexpectedly, we encountered substantial technical and biological noise including chimeric packaging products, necessitating development of novel techniques to accurately deconvolve enhancer expression patterns. These results underscore the need for improved methods to mitigate noise and highlight the complexity of enhancer AAV biology in vivo.