Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM
Name
Wong-2016-Multiplexed barcoded.pdf
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915.51 KB
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Checksum (MD5)
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Author(s)
Wong, Siu Lun
Cui, Cheryl
Pregernig, Gabriela
Milani, Pamela
Choi, Gigi C. G.
Adam, Miriam
Perli, Samuel D.
Kazer, Samuel Weisgurt
Gaillard de Saint Germain, Alethe
Hermann, Mario
Date Issued
February 2016
Journal
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences (U.S.)
Citation
Wong, Alan S. L. et al. “Multiplexed Barcoded CRISPR-Cas9 Screening Enabled by CombiGEM.” Proceedings of the National Academy of Sciences 113.9 (2016): 2544–2549. © 2016 National Academy of Sciences
Version
Final published version
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.
MIT Department
Massachusetts Institute of Technology. Synthetic Biology Center
Massachusetts Institute of Technology. Institute for Medical Engineering & Science
Massachusetts Institute of Technology. Department of Biological Engineering
Massachusetts Institute of Technology. Department of Chemistry
Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science
Massachusetts Institute of Technology. Research Laboratory of Electronics
Ragon Institute of MGH, MIT and Harvard
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Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.
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DOI of Published Version
https://doi.org/10.1073/pnas.1517883113
