Technical and biological sources of noise confound multiplexed enhancer AAV screening [dataset2]

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 perform barcoded multiplexed screening of enhancer AAVs at cell type resolution using single cell RNA sequencing and taxonomy mapping. We executed a proof-of-concept study using small pools of well-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. Overall design: Single-cell RNA-seq of flow cytometry-sorted H2B-YFP-expressing mouse brain nuclei. H2B-YFP expression was driven by cell type-specific enhancer-AAV PHP.eB vectors, injected at a moderate dose (5e11 vg/mouse) in young adulthood, and nuclei were prepared and sorted 3-4 weeks later. Nuclei from three regions were sorted: anterior lateral motor cortex (ALM) and striatum (STR) and primary visual cortex (VISp). Libraries were generated from one nucleus per library prep, performed using the SMART-Seq v4 procedure (Takara), and sequenced by paired-end sequencing at ~0.5M reads/nucleus on the Illumina platform.