title: Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM.
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.
authors: Synthetic Biology Group, Massachusetts Institute of Technology (MIT) Synthetic Biology Center, MIT, Cambridge, MA 02139; Research Laboratory of Electronics, MIT, Cambridge, MA 02139;
authors: Alan S L
authors: AS
authors: Wong
authors: Synthetic Biology Group, Massachusetts Institute of Technology (MIT) Synthetic Biology Center, MIT, Cambridge, MA 02139; Research Laboratory of Electronics, MIT, Cambridge, MA 02139;
authors: Gigi C G
authors: GC
authors: Choi
authors: Synthetic Biology Group, Massachusetts Institute of Technology (MIT) Synthetic Biology Center, MIT, Cambridge, MA 02139; Harvard University-MIT Division of Health Sciences and Technology, Cambridge, MA 02139;
authors: Cheryl H
authors: CH
authors: Cui
authors: Department of Biological Engineering, MIT, Cambridge, MA 02139;
authors: Gabriela
authors: G
authors: Pregernig
authors: Department of Biological Engineering, MIT, Cambridge, MA 02139;
authors: Pamela
authors: P
authors: Milani
authors: Department of Biological Engineering, MIT, Cambridge, MA 02139;
authors: Miriam
authors: M
authors: Adam
authors: Synthetic Biology Group, Massachusetts Institute of Technology (MIT) Synthetic Biology Center, MIT, Cambridge, MA 02139; Department of Electrical Engineering and Computer Science, MIT, Cambridge, MA 02139;
authors: Samuel D
authors: SD
authors: Perli
authors: Institute for Medical Engineering and Science and Department of Chemistry, MIT, Cambridge, MA 02139; Ragon Institute of Massachusetts General Hospital, MIT, and Harvard University, Cambridge, MA 02139; Broad Institute of MIT and Harvard University, Cambridge, MA 02142.
authors: Samuel W
authors: SW
authors: Kazer
authors: Institute for Medical Engineering and Science and Department of Chemistry, MIT, Cambridge, MA 02139; Ragon Institute of Massachusetts General Hospital, MIT, and Harvard University, Cambridge, MA 02139; Broad Institute of MIT and Harvard University, Cambridge, MA 02142.
authors: Aleth
authors: A
authors: Gaillard
authors: Synthetic Biology Group, Massachusetts Institute of Technology (MIT) Synthetic Biology Center, MIT, Cambridge, MA 02139; Research Laboratory of Electronics, MIT, Cambridge, MA 02139;
authors: Mario
authors: M
authors: Hermann
authors: Institute for Medical Engineering and Science and Department of Chemistry, MIT, Cambridge, MA 02139; Ragon Institute of Massachusetts General Hospital, MIT, and Harvard University, Cambridge, MA 02139; Broad Institute of MIT and Harvard University, Cambridge, MA 02142.
authors: Alex K
authors: AK
authors: Shalek
authors: Department of Biological Engineering, MIT, Cambridge, MA 02139;
authors: Ernest
authors: E
authors: Fraenkel
authors: Synthetic Biology Group, Massachusetts Institute of Technology (MIT) Synthetic Biology Center, MIT, Cambridge, MA 02139; Research Laboratory of Electronics, MIT, Cambridge, MA 02139; Department of Biological Engineering, MIT, Cambridge, MA 02139; Department of Electrical Engineering and Computer Science, MIT, Cambridge, MA 02139; timlu@mit.edu.
authors: Timothy K
authors: TK
authors: Lu
mesh_terms: D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D058893:DNA Barcoding, Taxonomic; D006801:Humans
keywords: CRISPR-Cas; CombiGEM; genetic perturbations; high-throughput screening; multifactorial genetics