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Edited by Jennifer A. Doudna, University of California, Berkeley, CA, and approved December 31, 2015 (received for review September 9, 2015)
Significance
The systematic discovery of new gene and drug combinations that modulate complex biological phenotypes and human diseases requires scalable and multiplexed screening technologies. We leverage the programmability of the CRISPR-Cas9 system for multiplexed targeting of specific genomic loci and the versatility of the combinatorial genetics en masse (CombiGEM) technology to rapidly assemble barcoded combinatorial genetic perturbation libraries that can be tracked with high-throughput sequencing. CombiGEM-CRISPR enables simple, massively parallel screening of barcoded combinatorial gene perturbations in human cells, and the translation of these hits into effective drug combinations. This approach is broadly applicable for performing pooled combinatorial genetic perturbations to map out how the orchestrated action of genes controls complex phenotypes and to translate these findings into novel drug combinations.
Abstract
The orchestrated action of genes controls complex biological phenotypes, yet the systematic discovery of gene and drug combinations that modulate these phenotypes in human cells is labor intensive and challenging to scale. Here, we created a platform for the massively parallel screening of barcoded combinatorial gene perturbations in human cells and translated these hits into effective drug combinations. This technology leverages the simplicity of the CRISPR-Cas9 system for multiplexed targeting of specific genomic loci and the versatility of combinatorial genetics en masse (CombiGEM) to rapidly assemble barcoded combinatorial genetic libraries that can be tracked with high-throughput sequencing. We applied CombiGEM-CRISPR to create a library of 23,409 barcoded dual guide-RNA (gRNA) combinations and then perform a high-throughput pooled screen to identify gene pairs that inhibited ovarian cancer cell growth when they were targeted. We validated the growth-inhibiting effects of specific gene sets, including epigenetic regulators KDM4C/BRD4 and KDM6B/BRD4, via individual assays with CRISPR-Cas–based knockouts and RNA-interference–based knockdowns. We also tested small-molecule drug pairs directed against our pairwise hits and showed that they exerted synergistic antiproliferative effects against ovarian cancer cells. We envision that the CombiGEM-CRISPR platform will be applicable to a broad range of biological settings and will accelerate the systematic identification of genetic combinations and their translation into novel drug combinations that modulate complex human disease phenotypes.
Footnotes
↵1A.S.L.W., G.C.G.C., and C.H.C. contributed equally to this work.
↵2Present address: School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong.
- ↵3To whom correspondence should be addressed. Email: timlu{at}mit.edu.
Author contributions: A.S.L.W., G.C.G.C., C.H.C., and T.K.L. designed research; A.S.L.W., G.C.G.C., C.H.C., P.M., M.A., S.W.K., A.G., and M.H. performed research; A.K.S. and E.F. contributed new reagents/analytic tools; A.S.L.W., G.C.G.C., C.H.C., G.P., S.D.P., and T.K.L. analyzed data; and A.S.L.W., G.C.G.C., C.H.C., and T.K.L. wrote the paper.
Conflict of interest statement: T.K.L., A.S.L.W., and G.C.G.C. have filed a patent application based on this work with the US Patent and Trademark Office.
This article is a PNAS Direct Submission.
Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE71074).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1517883113/-/DCSupplemental.
Freely available online through the PNAS open access option.
Multiplexed CRISPR-Cas9 screening by CombiGEM
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