Massively parallel in vivo Perturb-seq screening

Advances in genomics have identified thousands of risk genes impacting human health and diseases, but the functions of these genes and their mechanistic contribution to disease are often unclear. Moving beyond identification to actionable biological pathways requires dissecting risk gene function and cell type-specific action in intact tissues. This gap can in part be addressed by in vivo Perturb-seq, a method that combines state-of-the-art gene editing tools for programmable perturbation of genes with high-content, high-resolution single-cell genomic assays as phenotypic readouts. Here we describe a detailed protocol to perform massively parallel in vivo Perturb-seq using several versatile adeno-associated virus (AAV) vectors and provide guidance for conducting successful downstream analyses. Expertise in mouse work, AAV production and single-cell genomics is required. We discuss key parameters for designing in vivo Perturb-seq experiments across diverse biological questions and contexts. We further detail the step-by-step procedure, from designing a perturbation library to producing and administering AAV, highlighting where quality control checks can offer critical go-no-go points for this time- and cost-expensive method. Finally, we discuss data analysis options and available software. In vivo Perturb-seq has the potential to greatly accelerate functional genomics studies in mammalian systems, and this protocol will help others adopt it to answer a broad array of biological questions. From guide RNA design to tissue collection and data collection, this protocol is expected to take 9-15 weeks to complete, followed by data analysis.