Abstract
Spheroid culture systems have allowed in vitro propagation of cells unable to grow in canonical cell culturing conditions, and may capture cellular contexts that model tumor growth better than current model systems. The insights gleaned from genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening of thousands of cancer cell lines grown in conventional culture conditions illustrate the value of such CRISPR pooled screens. It is clear that similar genome-wide CRISPR screens of three-dimensional spheroid cultures will be important for future biological discovery. Here, we present a protocol for genome-wide CRISPR screening of three-dimensional neurospheres. While many in-depth protocols and discussions have been published for more typical cell lines, few detailed protocols are currently available in the literature for genome-wide screening in spheroidal cell lines. For those who want to screen such cell lines, and particularly neurospheres, we provide a step-by-step description of assay development tests to be performed before screening, as well as for the screen itself. We highlight considerations of variables that make these screens distinct from, or similar to, typical nonspheroid cell lines throughout. Finally, we illustrate typical outcomes of neurosphere genome-wide screens, and how neurosphere screens typically produce slightly more heterogeneous signal distributions than more canonical cancer cell lines. Completion of this entire protocol will take 8–12 weeks from the initial assay development tests to deconvolution of the sequencing data.
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Data availability
For the canonical cancer cell line DEL raw data associated with Fig. 4, see 22Q4 of the DepMap LFC’s https://depmap.org/portal/. For the mouse neurosphere cell line raw data associated with Fig. 4, see Supplementary Table 1.
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Acknowledgements
We thank and acknowledge the following funding sources: the Functional Genomics Consortium (D.E.R.), the DFCI Pediatric Low-Grade Glioma Program (P.B.), the Pediatric Brain Tumor Fund (P.B.) and the Broad Escape Velocity Award (P.B.). We also thank J. Dempster and I. Boyle for helping to identify a cell line with average null-normalized mean difference, GPP operations, GPP informatics, GPP screening, GPP production and GPP R&D for producing and creating optimal reagents and libraries.
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P.B., F.P., N.S.P. and D.E.R. conceived the project. A.B.G., Z.K., M.W., E.M.G., K.N.Z., K.Q., D.N., A.-L.C., P.B., F.P., N.S.P. and D.E.R. optimized the protocol and supervised and performed the screens and analyzed the data presented. All authors wrote, edited and approved the manuscript. P.B., F.P., N.S.P. and D.E.R. supervised the overall work.
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P.B. receives grant funding from Novartis Institute of Biomedical Research for an unrelated project and has received grant funding from Deerfield Therapeutics for unrelated work. She has served on a paid advisory panel for QED Therapeutics for unrelated work. D.E.R. receives research funding from members of the Functional Genomics Consortium (Abbvie, BMS, Jannsen, Merck, Vir), and is a director of Addgene, Inc. F.P. is a current employee of Merck Research Laboratories. N.S.P. is a current employee of Aera Therapeutics. All other authors declare no competing interests.
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Nature Protocols thanks Nobuaki Takahashi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key references related to this protocol
Khadka, P. et al. Nat. Commun. 13, 604 (2022): https://doi.org/10.1038/s41467-022-28198-8
Bandopadhayay, P. et al. Nat. Commun. 10, 2400 (2019): https://doi.org/10.1038/s41467-019-10307-9
Sanson, K. R. et al. Nat. Commun. 9, 5416 (2018): https://doi.org/10.1038/s41467-018-07901-8
Supplementary information
Supplementary Information
Supplementary Methods and Figs. 1 and 2.
Supplementary Table
Raw data for Fig. 4b
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Abid, T., Goodale, A.B., Kalani, Z. et al. Genome-wide pooled CRISPR screening in neurospheres. Nat Protoc 18, 2014–2031 (2023). https://doi.org/10.1038/s41596-023-00835-6
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DOI: https://doi.org/10.1038/s41596-023-00835-6
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