Technical and biological sources of noise confound multiplexed enhancer AAV screening [dataset4]

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: We injected young adult C57Bl/6J mice with various cell type-specific PHP.eB-encapsidated barcoded enhancer-AAV vectors, either alone or as pools. Three to four weeks later, we profiled YFP-expressing whole cells [or nuclei] from C57Bl/6 cell suspensions using fluorescence activated cell sorting (FACS). These sorted cells were then prepared for scRNA-seq using the 10X Chromium NextGEM v3.1 3' GEX droplet-based sc/snRNA-seq platform. cDNA and sequencing libraries were prepared according to the manufacturer's protocols, and then sequenced to a depth of approx 125k read pairs per cell (cDNA libraries). Additionally, in some experiments cDNA libraries were amplified with custom indexed PCR primers to maximize detection of AAV-encoded mRNA barcodes (mBCs). Furthermore, in some experiments low-molecular weight supernatant from cDNA bead purification was amplified with different custom indexed PCR primers to detect AAV-encoded Tornado barcodes (oBCs). GEX and mBC and oBC libraries were each sequenced.