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Pooled genetic perturbation screens with image-based phenotypes

Abstract

Discovery of the genetic components underpinning fundamental and disease-related processes is being rapidly accelerated by combining efficient, programmable genetic engineering with phenotypic readouts of high spatial, temporal and/or molecular resolution. Microscopy is a fundamental tool for studying cell biology, but its lack of high-throughput sequence readouts hinders integration in large-scale genetic screens. Optical pooled screens using in situ sequencing provide massively scalable integration of barcoded lentiviral libraries (e.g., CRISPR perturbation libraries) with high-content imaging assays, including dynamic processes in live cells. The protocol uses standard lentiviral vectors and molecular biology, providing single-cell resolution of phenotype and engineered genotype, scalability to millions of cells and accurate sequence reads sufficient to distinguish >106 perturbations. In situ amplification takes ~2 d, while sequencing can be performed in ~1.5 h per cycle. The image analysis pipeline provided enables fully parallel automated sequencing analysis using a cloud or cluster computing environment.

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Fig. 1: Pooled screening approaches and applications of optical pooled screens.
Fig. 2: Overview of optical pooled screening.
Fig. 3: Technical performance and quality control of SBS.
Fig. 4: Anticipated results.

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Data availability

The dataset used to produce Figs. 3 and 4 was originally published in ref. 45 and is publicly available from Cell-IDR69 (idr0071, experiment C). Source data are provided with this paper.

Code availability

The Python package and associated resources for the sgRNA library pool design and image analysis pipeline are freely available at https://github.com/feldman4/OpticalPooledScreens64 under the terms of the MIT license.

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Acknowledgements

The authors thank E. Botelho for research support; K. Caetano-Anolles for critical reading of the manuscript; R. Walton for assistance with figures; Pulse Media Inc. for producing the video; A. Garrity, J. Schmid-Burgk and A. Mezger for contributions to the development of the protocol; I. Cheeseman, J.T. Neal, A. Carpenter, F. Zhang and A. Regev for helpful discussions; and all members of the Blainey Lab for continued collaboration and support. This work was supported by two grants from the National Human Genome Research Institute (HG009283 and HG006193). L.F. was supported by a National Defense Science and Engineering Graduate Fellowship. R.C. was supported by a Fannie and John Hertz Foundation Fellowship and an NSF Graduate Research Fellowship.

Author information

Authors and Affiliations

Authors

Contributions

D.F., R.C. and A.S. developed the optical pooled screening protocol and performed experiments. D.F. and L.F. analyzed the data and created the accompanying code repository. D.F., L.F., A.L., R.C., F.T., A.S. and P.C.B. wrote the manuscript. M.D.L., B.S. and A.S. made the video.

Corresponding author

Correspondence to Paul C. Blainey.

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Competing interests

P.C.B. is a consultant to and/or equity holder in companies in the life sciences industries, including 10X Genomics, GALT, Celsius Therapeutics, Next Generation Diagnostics, Cache DNA and Concerto Biosciences. P.C.B.’s laboratory receives research funding from Calico Life Sciences and Merck for work related to genetic screening. The Broad Institute and MIT have filed US patent applications on work described here and may seek to license the technology.

Additional information

Peer review information Nature Protocols thanks Je H. Lee, Ophir Shalem and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Funk, L. et al. Preprint at bioRxiv (2021): https://doi.org/10.1101/2021.11.28.470116

Feldman, D. et al. Cell 179, 787–799.e17 (2019): https://doi.org/10.1016/j.cell.2019.09.016

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2.

Supplementary Tables

Supplementary Table 1. Spreadsheet calculator for estimating the scale of an optical pooled screen. Supplementary Table 2. Oligo pool dialout primer sequences. Supplementary Table 3. Primer sequences for NGS library preparation. Supplementary Table 4. Oligo sequences for in situ amplification of sgRNA sequences.

Source data

Source Data Fig. 3

Source tabular data for Fig. 3b–e (separate sheet/tab for each panel)

Source Data Fig. 4

Source tabular data for Fig. 4b,c (separate sheet/tab for each panel)

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Feldman, D., Funk, L., Le, A. et al. Pooled genetic perturbation screens with image-based phenotypes. Nat Protoc 17, 476–512 (2022). https://doi.org/10.1038/s41596-021-00653-8

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