Skip to main content

Main menu

  • Home
  • Articles
    • Current
    • Special Feature Articles - Most Recent
    • Special Features
    • Colloquia
    • Collected Articles
    • PNAS Classics
    • List of Issues
  • Front Matter
    • Front Matter Portal
    • Journal Club
  • News
    • For the Press
    • This Week In PNAS
    • PNAS in the News
  • Podcasts
  • Authors
    • Information for Authors
    • Editorial and Journal Policies
    • Submission Procedures
    • Fees and Licenses
  • Submit
  • Submit
  • About
    • Editorial Board
    • PNAS Staff
    • FAQ
    • Accessibility Statement
    • Rights and Permissions
    • Site Map
  • Contact
  • Journal Club
  • Subscribe
    • Subscription Rates
    • Subscriptions FAQ
    • Open Access
    • Recommend PNAS to Your Librarian

User menu

  • Log in
  • My Cart

Search

  • Advanced search
Home
Home
  • Log in
  • My Cart

Advanced Search

  • Home
  • Articles
    • Current
    • Special Feature Articles - Most Recent
    • Special Features
    • Colloquia
    • Collected Articles
    • PNAS Classics
    • List of Issues
  • Front Matter
    • Front Matter Portal
    • Journal Club
  • News
    • For the Press
    • This Week In PNAS
    • PNAS in the News
  • Podcasts
  • Authors
    • Information for Authors
    • Editorial and Journal Policies
    • Submission Procedures
    • Fees and Licenses
  • Submit
Research Article

Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM

Alan S. L. Wong, Gigi C. G. Choi, Cheryl H. Cui, Gabriela Pregernig, Pamela Milani, Miriam Adam, Samuel D. Perli, Samuel W. Kazer, Aleth Gaillard, Mario Hermann, Alex K. Shalek, Ernest Fraenkel, and Timothy K. Lu
  1. aSynthetic Biology Group, Massachusetts Institute of Technology (MIT) Synthetic Biology Center, MIT, Cambridge, MA 02139;
  2. bResearch Laboratory of Electronics, MIT, Cambridge, MA 02139;
  3. cHarvard University–MIT Division of Health Sciences and Technology, Cambridge, MA 02139;
  4. dDepartment of Biological Engineering, MIT, Cambridge, MA 02139;
  5. eDepartment of Electrical Engineering and Computer Science, MIT, Cambridge, MA 02139;
  6. fInstitute for Medical Engineering and Science and Department of Chemistry, MIT, Cambridge, MA 02139;
  7. gRagon Institute of Massachusetts General Hospital, MIT, and Harvard University, Cambridge, MA 02139;
  8. hBroad Institute of MIT and Harvard University, Cambridge, MA 02142

See allHide authors and affiliations

PNAS March 1, 2016 113 (9) 2544-2549; first published February 10, 2016; https://doi.org/10.1073/pnas.1517883113
Alan S. L. Wong
aSynthetic Biology Group, Massachusetts Institute of Technology (MIT) Synthetic Biology Center, MIT, Cambridge, MA 02139;
bResearch Laboratory of Electronics, MIT, Cambridge, MA 02139;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Gigi C. G. Choi
aSynthetic Biology Group, Massachusetts Institute of Technology (MIT) Synthetic Biology Center, MIT, Cambridge, MA 02139;
bResearch Laboratory of Electronics, MIT, Cambridge, MA 02139;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Cheryl H. Cui
aSynthetic Biology Group, Massachusetts Institute of Technology (MIT) Synthetic Biology Center, MIT, Cambridge, MA 02139;
cHarvard University–MIT Division of Health Sciences and Technology, Cambridge, MA 02139;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Gabriela Pregernig
dDepartment of Biological Engineering, MIT, Cambridge, MA 02139;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Pamela Milani
dDepartment of Biological Engineering, MIT, Cambridge, MA 02139;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Miriam Adam
dDepartment of Biological Engineering, MIT, Cambridge, MA 02139;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Samuel D. Perli
aSynthetic Biology Group, Massachusetts Institute of Technology (MIT) Synthetic Biology Center, MIT, Cambridge, MA 02139;
eDepartment of Electrical Engineering and Computer Science, MIT, Cambridge, MA 02139;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Samuel W. Kazer
fInstitute for Medical Engineering and Science and Department of Chemistry, MIT, Cambridge, MA 02139;
gRagon Institute of Massachusetts General Hospital, MIT, and Harvard University, Cambridge, MA 02139;
hBroad Institute of MIT and Harvard University, Cambridge, MA 02142
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Aleth Gaillard
fInstitute for Medical Engineering and Science and Department of Chemistry, MIT, Cambridge, MA 02139;
gRagon Institute of Massachusetts General Hospital, MIT, and Harvard University, Cambridge, MA 02139;
hBroad Institute of MIT and Harvard University, Cambridge, MA 02142
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Mario Hermann
aSynthetic Biology Group, Massachusetts Institute of Technology (MIT) Synthetic Biology Center, MIT, Cambridge, MA 02139;
bResearch Laboratory of Electronics, MIT, Cambridge, MA 02139;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Alex K. Shalek
fInstitute for Medical Engineering and Science and Department of Chemistry, MIT, Cambridge, MA 02139;
gRagon Institute of Massachusetts General Hospital, MIT, and Harvard University, Cambridge, MA 02139;
hBroad Institute of MIT and Harvard University, Cambridge, MA 02142
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ernest Fraenkel
dDepartment of Biological Engineering, MIT, Cambridge, MA 02139;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Timothy K. Lu
aSynthetic Biology Group, Massachusetts Institute of Technology (MIT) Synthetic Biology Center, MIT, Cambridge, MA 02139;
bResearch Laboratory of Electronics, MIT, Cambridge, MA 02139;
dDepartment of Biological Engineering, MIT, Cambridge, MA 02139;
eDepartment of Electrical Engineering and Computer Science, MIT, Cambridge, MA 02139;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: timlu@mit.edu
  1. Edited by Jennifer A. Doudna, University of California, Berkeley, CA, and approved December 31, 2015 (received for review September 9, 2015)

  • Article
  • Figures & SI
  • Info & Metrics
  • PDF
Loading

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.

  • CRISPR-Cas
  • CombiGEM
  • multifactorial genetics
  • genetic perturbations
  • high-throughput screening

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.

View Full Text
PreviousNext
Back to top
Article Alerts
Email Article

Thank you for your interest in spreading the word on PNAS.

NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

Enter multiple addresses on separate lines or separate them with commas.
Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM
(Your Name) has sent you a message from PNAS
(Your Name) thought you would like to see the PNAS web site.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Citation Tools
Multiplexed CRISPR-Cas9 screening by CombiGEM
Alan S. L. Wong, Gigi C. G. Choi, Cheryl H. Cui, Gabriela Pregernig, Pamela Milani, Miriam Adam, Samuel D. Perli, Samuel W. Kazer, Aleth Gaillard, Mario Hermann, Alex K. Shalek, Ernest Fraenkel, Timothy K. Lu
Proceedings of the National Academy of Sciences Mar 2016, 113 (9) 2544-2549; DOI: 10.1073/pnas.1517883113

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Request Permissions
Share
Multiplexed CRISPR-Cas9 screening by CombiGEM
Alan S. L. Wong, Gigi C. G. Choi, Cheryl H. Cui, Gabriela Pregernig, Pamela Milani, Miriam Adam, Samuel D. Perli, Samuel W. Kazer, Aleth Gaillard, Mario Hermann, Alex K. Shalek, Ernest Fraenkel, Timothy K. Lu
Proceedings of the National Academy of Sciences Mar 2016, 113 (9) 2544-2549; DOI: 10.1073/pnas.1517883113
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Mendeley logo Mendeley

Article Classifications

  • Biological Sciences
  • Systems Biology
Proceedings of the National Academy of Sciences: 113 (9)
Table of Contents

Submit

Sign up for Article Alerts

Jump to section

  • Article
    • Abstract
    • Results
    • Discussion
    • Materials and Methods
    • Acknowledgments
    • Footnotes
    • References
  • Figures & SI
  • Info & Metrics
  • PDF

You May Also be Interested in

Smoke emanates from Japan’s Fukushima nuclear power plant a few days after tsunami damage
Core Concept: Muography offers a new way to see inside a multitude of objects
Muons penetrate much further than X-rays, they do essentially zero damage, and they are provided for free by the cosmos.
Image credit: Science Source/Digital Globe.
Water from a faucet fills a glass.
News Feature: How “forever chemicals” might impair the immune system
Researchers are exploring whether these ubiquitous fluorinated molecules might worsen infections or hamper vaccine effectiveness.
Image credit: Shutterstock/Dmitry Naumov.
Venus flytrap captures a fly.
Journal Club: Venus flytrap mechanism could shed light on how plants sense touch
One protein seems to play a key role in touch sensitivity for flytraps and other meat-eating plants.
Image credit: Shutterstock/Kuttelvaserova Stuchelova.
Illustration of groups of people chatting
Exploring the length of human conversations
Adam Mastroianni and Daniel Gilbert explore why conversations almost never end when people want them to.
Listen
Past PodcastsSubscribe
Panda bear hanging in a tree
How horse manure helps giant pandas tolerate cold
A study finds that giant pandas roll in horse manure to increase their cold tolerance.
Image credit: Fuwen Wei.

Similar Articles

Site Logo
Powered by HighWire
  • Submit Manuscript
  • Twitter
  • Facebook
  • RSS Feeds
  • Email Alerts

Articles

  • Current Issue
  • Special Feature Articles – Most Recent
  • List of Issues

PNAS Portals

  • Anthropology
  • Chemistry
  • Classics
  • Front Matter
  • Physics
  • Sustainability Science
  • Teaching Resources

Information

  • Authors
  • Editorial Board
  • Reviewers
  • Subscribers
  • Librarians
  • Press
  • Cozzarelli Prize
  • Site Map
  • PNAS Updates
  • FAQs
  • Accessibility Statement
  • Rights & Permissions
  • About
  • Contact

Feedback    Privacy/Legal

Copyright © 2021 National Academy of Sciences. Online ISSN 1091-6490