CRISPR-StAR enables high-resolution genetic screening in complex in vivo models
Pooled genetic screening with CRISPR-Cas9 has enabled genome-wide, high-resolution mapping of genes to phenotypes, but assessing the effect of a given genetic perturbation requires evaluation of each single guide RNA (sgRNA) in hundreds of cells to counter stochastic genetic drift and obtain robust results. However, resolution is limited in complex, heterogeneous models, such as organoids or tumors transplanted into mice, because achieving sufficient representation requires impractical scaling. This is due to bottleneck effects and biological heterogeneity of cell populations. Here we introduce CRISPR-StAR, a screening method that uses internal controls generated by activating sgRNAs in only half the progeny of each cell subsequent to re-expansion of the cell clone. Our method overcomes both intrinsic and extrinsic heterogeneity as well as genetic drift in bottlenecks by generating clonal, single-cell-derived intrinsic controls. We use CRISPR-StAR to identify in-vivo-specific genetic dependencies in a genome-wide screen in mouse melanoma. Benchmarking against conventional screening demonstrates the improved data quality provided by this technology.
Authors
- Gintautas Vainorius (ORCID: https://orcid.org/0000-0001-6552-4743)
- Juliane Christina Baar (ORCID: https://orcid.org/0000-0002-6949-282X)
- Anais Elewaut
- Maria Novatchkova (ORCID: https://orcid.org/0000-0002-8462-7171)
- Esther C. H. Uijttewaal (ORCID: https://orcid.org/0000-0003-3892-2820)
- Daniel Schramek (ORCID: https://orcid.org/0000-0001-9977-2104)
- S. Leij (ORCID: https://orcid.org/0009-0008-4349-2806)
- Ulrich Elling (ORCID: https://orcid.org/0000-0002-4514-2809)
- Annika Charlotte Sell (ORCID: https://orcid.org/0009-0006-2272-0094)
- Christopher Lowden (ORCID: https://orcid.org/0000-0002-1839-164X)
- Joonsun Lee (ORCID: https://orcid.org/0009-0004-5135-1430)
- Jiaye Yang
- Anna C. Obenauf (ORCID: https://orcid.org/0000-0002-9556-5914)
- Milanka Gavrilovic
- Julia Sinner
- Naomi Botay
- Tobias Potzler
Institutions
- Institute of Molecular Biotechnology (AT)
- Mount Sinai Hospital (CA)
- University of Toronto (CA)
- Research Institute of Molecular Pathology (AT)
- Lunenfeld-Tanenbaum Research Institute (CA)
- Wiener Krankenanstaltenverbund (AT)
- Vienna Biocenter (AT)
- Medical University of Vienna (AT)
Publication Details
- Journal
- Nature Biotechnology
- Published
- 2024-12-16
- DOI
- https://doi.org/10.1038/s41587-024-02512-9
- Citations
- 18
- Primary Topic
- CRISPR and Genetic Engineering
- Type
- article
- Field-Weighted Citation Impact
- 4.21
Funders
- Canada Research Chairs
- Österreichischen Akademie der Wissenschaften
- Krembil Foundation
- Terry Fox Research Institute
- Staatssekretariat für Bildung, Forschung und Innovation