Genetic interaction mapping in mammalian cells using CRISPR interference

Abstract

We describe a combinatorial CRISPR interference (CRISPRi) screening platform for mapping genetic interactions in mammalian cells. We targeted 107 chromatin-regulation factors in human cells with pools of either single or double single guide RNAs (sgRNAs) to downregulate individual genes or gene pairs, respectively. Relative enrichment analysis of individual sgRNAs or sgRNA pairs allowed for quantitative characterization of genetic interactions, and comparison with protein–protein-interaction data revealed a functional map of chromatin regulation.

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Figure 1: The CRISPRi screening platform for studying genetic interactions.
Figure 2: Module map of chromatin-related genes, based on a curated set of the indicated protein complexes.

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Acknowledgements

The authors thank the members of the laboratories of L.S.Q. and N.J.K. for advice and helpful discussions, and the Stanford Functional Genomics Facility and UCSD Sequencing Facility for technical support. We thank D. Zhao from the laboratory of L.S.Q. for advice and help in cloning the sgRNA library. L.S.Q. acknowledges support from the NIH Office of the Director (OD), the National Institute of Dental & Craniofacial Research (NIDCR), the Department of Defense Breast Cancer Research Breakthrough Program, the Pew Charitable Trusts and the Alfred P. Sloan Foundation. D.D. and A.R. acknowledge support from the Quantitative Bioscience Institute (QBI). M.C., N.J.K. and L.S.Q. acknowledge support from the J. David Gladstone Biofulcrum Program. This work was supported by DP5 OD017887, NIH R01 DA036858, NIH U01EB021240, DOD W81XWH-17-1-0018, a Pew Scholar Fellowship and an Alfred P. Sloan Fellowship to L.S.Q., as well as by NIH grants P50 GM082250, U19 AI106754, P01 HL089707, R01 GM084279, U19 AI118610 and R01 AI120694 to N.J.K.

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Contributions

L.S.Q. and N.J.K. conceived the project. L.S.Q., N.J.K., D.D. and A.R. designed the experiments; L.S.Q. and D.D. designed the sgRNA library; D.D. constructed the sgRNA libraries and performed library screens; D.D. and A.R. performed sample preparation and sequencing; A.R. analyzed the sequencing data, curated the data sets and scored the data; D.D., S.-H.C. and M.C. performed the CRISPRi validation experiments; D.E.G. performed the esiRNA validation experiments; M.S. contributed to analysis and figure generation; J.P.S., T.I. and P.M. provided technical advice; L.S.Q., N.J.K. and A.R. wrote the paper.

Corresponding authors

Correspondence to Lei S Qi or Nevan J Krogan.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Tables 1–8, and Supplementary Notes 1 and 2 (PDF 28438 kb)

Supplementary Data Set 1

Genetic interactions used to build networks on Figure 2a,b (TXT 5 kb)

Supplementary Data Set 2

Single sgRNA screens and rapamycin screen raw data (XLSX 70 kb)

Supplementary Data Set 3

Double sgRNA screens raw data and data used in individual figures (ZIP 9443 kb)

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Du, D., Roguev, A., Gordon, D. et al. Genetic interaction mapping in mammalian cells using CRISPR interference. Nat Methods 14, 577–580 (2017). https://doi.org/10.1038/nmeth.4286

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