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Massively parallel in vivo Perturb-seq screening

Nature Protocols volume 20, pages 1733–1767 (2025)Cite this article

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Abstract

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.

Key points

  • Versatile adeno-associated virus (AAV) vectors are combined with a transposon system to achieve high-level fast-onset expression of guide RNA perturbation libraries in live animals.

  • This protocol outlines strategies for designing the perturbation library, producing and administering AAV and performing downstream single-cell RNA sequencing and computational analyses.

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Fig. 1: Overview of massively parallel in vivo Perturb-seq screening.
Fig. 2: Schematics of the molecular design to enhance transgene expression through integrations.
Fig. 3: Examples of evaluation of in vivo administration density and multiplexity of AAV vectors before conducting a massively parallel in vivo Perturb-seq experiment.
Fig. 4: QC in a massively parallel in vivo Perturb-seq experiment.
Fig. 5: Anticipated result from massively parallel in vivo Perturb-seq.
Fig. 6: Example image of streaks made on an LB agar plate using a multichannel pipette after an overnight incubation.

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Data availability

The data included in this protocol are from the key reference publication11. The raw data generated in that key reference study are available on Mendeley Data (https://doi.org/10.17632/hvb39r62xw.1), NCBI Gene Expression Omnibus (GEO: GSE249416) and the Broad single cell portal (https://singlecell.broadinstitute.org/single_cell/study/SCP2443).

Code availability

The analysis pipeline used in the key reference publication is deposited in the GitHub repository (https://github.com/jinlabneurogenomics). The Python scripts referenced in this protocol are available in the Supplementary Information (Supplementary Softwares 1 and 2).

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Acknowledgements

We thank B. Wu for advice on the scRNA-seq experiments; the Department of Animal Resources, Genomics Core and Flow Cytometry Core facilities at Scripps Research for technical assistance; N. Huynh, S. Simmons, B. Wu, J. Li, A. Selstad and E. Petty for comments on the manuscript; and all members of the Jin lab for their help and support. X.Z. and P.C.T. were supported by the Dorris Scholar Award. X.Z. was supported by Frank J. Dixon Graduate Fellowship. C.M.W. was supported by Skaggs-Oxford Fellowship and The Schimmel Family Endowed Fellowship. X.J. and this work were supported by the Simons Foundation for Autism Research Initiative Collaboration on Sex Differences (SFARI 736613), National Institute of Health (R01HG012819, R01MH137042), Impetus grant, One Mind Rising Star Award, Klingenstein-Simons Fellowship Award, G. Harold and Leila Y. Mathers Foundation, Larry L. Hillblom Foundation, Scripps Collaborative Innovative Fund, Chan Zuckerberg Initiative, Conrad Prebys Foundation, Pew Charitable Trusts, McKnight Foundation, Astera Institute and James Fickel.

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Authors and Affiliations

  1. Department of Neuroscience, Dorris Neuroscience Center, Scripps Research, La Jolla, CA, USA

    Xinhe Zheng, Patrick C. Thompson, Cassandra M. White & Xin Jin

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X.Z., P.C.T., C.M.W. and X.J. designed and performed the experiments and wrote the manuscript.

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Correspondence to Xin Jin.

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X.J. and X.Z. are co-inventors on in vivo AAV-based Perturb-seq and CRISPR inventions filed by Scripps Research relating to the work in this protocol. P.C.T. and C.M.W. declare no competing interests.

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Key reference

Zheng, X. et al. Cell 187, 3236–3248.e21 (2024): https://doi.org/10.1016/j.cell.2024.04.050

Extended data

Extended Data Fig. 1 FACS analysis and gating strategy for identifying BFP+ perturbed cells.

Dissociated cortical cell suspension from P7 mouse brains was stained with Vybrant™ DyeCycle™ Ruby and analyzed by FACS. First, cells are identified based on size (forward scatter, FSC-A) versus granularity (side scatter, SSC-A). Single cells were isolated by excluding doublets using SSC-A versus SSC-H. Then, a Vybrant (cell-permeable stain) versus FSC-A gate was used to remove cytosolic debris (Vybrant−). Alternatively, a viability dye (e.g., Sytox™ Red) can be used to exclude dead cells (SytoxRed+, not shown). Finally, the perturbed BFP+ population is defined by GFP (expressed constitutively in a Cas9 transgenic mouse line) versus mtagBFP (expressed in transduced cells) plot.

Supplementary information

Reporting Summary (download PDF )

Supplementary Table 1 (download XLSX )

AAV titration calculator.

Supplementary Software 1 and 2 (download ZIP )

2 Python scripts for generate gRNA oligo and map gRNA in fastq files separately.

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Zheng, X., Thompson, P.C., White, C.M. et al. Massively parallel in vivo Perturb-seq screening. Nat Protoc 20, 1733–1767 (2025). https://doi.org/10.1038/s41596-024-01119-3

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