Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM
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...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | en_US |
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National Academy of Sciences (U.S.)
2017
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| Online Access: | http://hdl.handle.net/1721.1/106350 https://orcid.org/0000-0001-5744-0873 https://orcid.org/0000-0002-0160-8742 https://orcid.org/0000-0001-7284-6337 https://orcid.org/0000-0003-0250-0474 https://orcid.org/0000-0002-8346-2184 https://orcid.org/0000-0002-7380-9594 https://orcid.org/0000-0001-9179-7972 https://orcid.org/0000-0001-9249-8181 https://orcid.org/0000-0002-9999-6690 |
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| author | 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 Shalek, Alexander K Fraenkel, Ernest Lu, Timothy K |
| author2 | Massachusetts Institute of Technology. Synthetic Biology Center |
| author_facet | Massachusetts Institute of Technology. Synthetic Biology Center 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 Shalek, Alexander K Fraenkel, Ernest Lu, Timothy K |
| author_sort | Wong, Siu Lun |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T11:08:26Z |
| format | Article |
| id | mit-1721.1/106350 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T11:08:26Z |
| publishDate | 2017 |
| publisher | National Academy of Sciences (U.S.) |
| record_format | dspace |
| spelling | mit-1721.1/1063502024-03-20T20:27:31Z Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM 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 Shalek, Alexander K Fraenkel, Ernest Lu, Timothy K 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 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 Shalek, Alexander K Fraenkel, Ernest Lu, Timothy K 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. National Institutes of Health (U.S.) (Grants DP2OD008435, P50GM098792, and R01 NS089076) United States. Office of Naval Research (Grant N00014-13-1-0424) United States. Defense Threat Reduction Agency Lawrence Ellison Foundation (New Scholar in Aging Award) Croucher Foundation Natural Sciences and Engineering Research Council of Canada (Postdoctoral Fellowship) 2017-01-12T14:34:58Z 2017-01-12T14:34:58Z 2016-02 2015-09 Article http://purl.org/eprint/type/JournalArticle 0027-8424 1091-6490 http://hdl.handle.net/1721.1/106350 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 https://orcid.org/0000-0001-5744-0873 https://orcid.org/0000-0002-0160-8742 https://orcid.org/0000-0001-7284-6337 https://orcid.org/0000-0003-0250-0474 https://orcid.org/0000-0002-8346-2184 https://orcid.org/0000-0002-7380-9594 https://orcid.org/0000-0001-9179-7972 https://orcid.org/0000-0001-9249-8181 https://orcid.org/0000-0002-9999-6690 en_US http://dx.doi.org/10.1073/pnas.1517883113 Proceedings of the National Academy of Sciences 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. application/pdf National Academy of Sciences (U.S.) PNAS |
| spellingShingle | 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 Shalek, Alexander K Fraenkel, Ernest Lu, Timothy K Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM |
| title | Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM |
| title_full | Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM |
| title_fullStr | Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM |
| title_full_unstemmed | Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM |
| title_short | Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM |
| title_sort | multiplexed barcoded crispr cas9 screening enabled by combigem |
| url | http://hdl.handle.net/1721.1/106350 https://orcid.org/0000-0001-5744-0873 https://orcid.org/0000-0002-0160-8742 https://orcid.org/0000-0001-7284-6337 https://orcid.org/0000-0003-0250-0474 https://orcid.org/0000-0002-8346-2184 https://orcid.org/0000-0002-7380-9594 https://orcid.org/0000-0001-9179-7972 https://orcid.org/0000-0001-9249-8181 https://orcid.org/0000-0002-9999-6690 |
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