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Synergistic drug combinations for cancer identified in a CRISPR screen for pairwise genetic interactions

Nature Biotechnology volume 35, pages 463474 (2017) | Download Citation

Abstract

Identification of effective combination therapies is critical to address the emergence of drug-resistant cancers, but direct screening of all possible drug combinations is infeasible. Here we introduce a CRISPR-based double knockout (CDKO) system that improves the efficiency of combinatorial genetic screening using an effective strategy for cloning and sequencing paired single guide RNA (sgRNA) libraries and a robust statistical scoring method for calculating genetic interactions (GIs) from CRISPR-deleted gene pairs. We applied CDKO to generate a large-scale human GI map, comprising 490,000 double-sgRNAs directed against 21,321 pairs of drug targets in K562 leukemia cells and identified synthetic lethal drug target pairs for which corresponding drugs exhibit synergistic killing. These included the BCL2L1 and MCL1 combination, which was also effective in imatinib-resistant cells. We further validated this system by identifying known and previously unidentified GIs between modifiers of ricin toxicity. This work provides an effective strategy to screen synergistic drug combinations in high-throughput and a CRISPR-based tool to dissect functional GI networks.

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Acknowledgements

We thank S. Collins and members of the Bassik laboratory for helpful discussions and critical reading of the manuscript, A. Sockell and L. Tonkin for technical assistance in deep sequencing, and M. Porteus and S. Mantri for CD34+ HSPCs. We thank M. Cleary, J. Duque-Afonso, and P. Jackson for helpful discussions regarding drug combination assays. We thank M. Kampmann, M. Horlbeck, L. Gilbert, and J. Weissman for helpful discussions, and L. Bruhn, C. Carstens, P. Sheffield, and B. Borgo of Agilent technologies for oligonucleotide synthesis and helpful discussions. GM12892 cells were a gift from S.B. Montgomery. The work was funded by the NIH Director's New Innovator Award Program (project no. 1DP2HD084069-01) NIH/NHGRI (training grant T32 HG000044 to D.W.M.), NIH/NCI 1U01CA199261-02, and a seed grant from Stanford ChEM-H. K.H. is supported by the Walter V. and Idun Berry award. E.E.J. was supported by the NIH (2T32CA009302) and a Hubert Shaw and Sandra Lui Stanford Graduate Fellowship.

Author information

Author notes

    • Kyuho Han
    •  & Edwin E Jeng

    These authors contributed equally to this work.

Affiliations

  1. Department of Genetics, Stanford University, Stanford, California, USA.

    • Kyuho Han
    • , Edwin E Jeng
    • , Gaelen T Hess
    • , David W Morgens
    • , Amy Li
    •  & Michael C Bassik
  2. Program in Cancer Biology, Stanford University, Stanford, California, USA.

    • Edwin E Jeng
  3. Chemistry, Engineering, and Medicine for Human Health (ChEM-H), Stanford University, Stanford, California, USA.

    • Michael C Bassik

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Contributions

K.H. and M.C.B. conceived and designed the study. K.H. designed the CDKO system and the scoring systems for GI map. K.H. analyzed the screen data and performed the GI and PPI analyses. K.H., A.L., and E.E.J. performed the CDKO screens. G.T.H., A.L., and D.W.M. performed the genome-wide screens for ricin modulators. D.W.M. selected the best-working sgRNAs to design the CDKO libraries. K.H. and E.E.J. validated the hits from the CDKO screens. E.E.J. performed the drug validations and related experiments. K.H., E.E.J., and M.C.B. wrote the manuscript. All authors discussed the results and the manuscript. M.C.B. supervised the study.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Michael C Bassik.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–13 and Supplementary Text

Excel files

  1. 1.

    Supplementary Table 1

    Selected 207 genes for DrugTarget-CDKO library

  2. 2.

    Supplementary Table 2

    700 sgRNAs for DrugTarget-CDKO library

  3. 3.

    Supplementary Table 3

    Distribution of double-sgRNAs per gene pair after filtering

  4. 4.

    Supplementary Table 4

    GI scores of DrugTarget-CDKO screen

  5. 5.

    Supplementary Table 5

    Selected 79 genes for Ricin-CDKO library

  6. 6.

    Supplementary Table 6

    284 sgRNAs for Ricin-CDKO library

  7. 7.

    Supplementary Table 7

    GI scores of Ricin-CDKO screen

  8. 8.

    Supplementary Table 8

    246 STRING interactions between 79 Ricin hits

  9. 9.

    Supplementary Table 9

    STRING interactions in the 66 most correlated gene pairs

  10. 10.

    Supplementary Table 10

    Selected 79 genes for DrugTarget Batch retest

  11. 11.

    Supplementary Table 11

    287 sgRNAs for DrugTarget Batch retest

  12. 12.

    Supplementary Table 12

    sgRNAs used for the validation of individual sgRNA pairs

  13. 13.

    Supplementary Table 13

    Summary of sgRNA and drug validations

  14. 14.

    Supplementary Table 14

    30 most synergistic DrugTarget pairs

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DOI

https://doi.org/10.1038/nbt.3834

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