CRISPR screening by AAV episome-sequencing (CrAAVe-seq): A scalable cell type-specific in vivo platform uncovers neuronal essential genes

There is a substantial need for scalable CRISPR-based genetic screening methods that can be applied in mammalian tissues in vivo while enablingcell-type-specific analysis. Here we developed an adeno-associated virus (AAV)-based CRISPR screening platform, CrAAVe-seq, that incorporates a Cre-sensitive sgRNA construct for pooled screening within targeted cell populations in mouse tissues. We used this approach to screen two large sgRNA libraries, which collectively target over 5,000 genes, in mouse brains and uncovered genes essential for neuronal survival, of which we validated Rabggta and Hspa5. We highlight the reproducibility and scalability of the platform and show that it is sufficiently sensitive for screening in a restricted subset of neurons. We systematically characterize the impact of sgRNA library size, mouse cohort size, the size of the targeted cell population, viral titer, and coinfection rate on screen performance to establish general guidelines for large-sca..., Animals
All mice were maintained according to the National Institutes of Health guidelines and all procedures used in this study were approved by the UCSF Institutional Animal Care and Use Committee. Mice were housed on a 12-h light/dark cycle at 22-25 °C, 50-60% humidity, and had food and water provided ad libitum. Mice were randomly assigned for the experimental groups at time of injection and both male and female mice were used. In accordance with approved protocol, mice were monitored post injection and if signs of distress appeared, mice were documented and euthanized promptly. The mice used in this study are LSL-dCas9-KRAB (LSL-CRISPRi) mice (B6;129S6-Gt(ROSA)26Sortm2(CAG-cas9*/ZNF10*)Gers/J, RRID: IMSR_JAX:033066)17 and dCas9-KRAB mice (B6.Cg-Igs2tm1(CAG-mCherry,-cas9/ZNF10*)Mtm/J, RRID: IMSR_JAX:030000). A summary of the individual mice used for CRISPR screening and select in vivo experiments is provided in Supplementary Table 1.
Plasmids
The screening vector pAP215 is shown in ..., , # Data from: CRISPR screening by AAV episome-sequencing (CrAAVe-seq): A scalable cell type-specific in vivo platform uncovers neuronal essential genes

[https://doi.org/10.5061/dryad.0k6djhb9t](https://doi.org/10.5061/dryad.0k6djhb9t)

## Description of the data and file structure

### Dataset Overview

**CRISPR Screen FASTQs & Image Data.** This dataset contains the raw FASTQ files from in vivo CRISPR screens performed using CrAAVe-seq on several populations of neurons in the mouse brain (see BioRxiv for further details: [https://doi.org/10.1101/2023.06.13.544831](https://doi.org/10.1101/2023.06.13.544831)). The datasets listed here are also described in Supplementary Table 1 (on BioRxiv). Each file represents a CRISPR screen in an individual mouse, except for when an \"inverted\" and \"total\" screen is conducted using PCR material from the same mouse, as noted below. Data files from *Hspa5* and *Rabggta* validation experiments are also included, both raw .tiff files and processed data fr...,