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Updated: Mar 26, 2026
Pooled CRISPR-Based Genetic Screens in Mammalian Cells
Published on: September 4, 2019
Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM
Alan S L Wong1, Gigi C G Choi1, Cheryl H Cui2
1Synthetic Biology Group, Massachusetts Institute of Technology (MIT) Synthetic Biology Center, MIT, Cambridge, MA 02139; Research Laboratory of Electronics, MIT, Cambridge, MA 02139;
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.
Insights
This study introduces CombiGEM-CRISPR, a platform for screening gene combinations to find effective drug pairs. It accelerates the discovery of genetic and drug combinations for human diseases.
Area of Science:
- Genomics
- Cancer Biology
- Drug Discovery
Background:
- Discovering gene and drug combinations to control cell phenotypes is challenging and difficult to scale.
- Systematic identification of genetic interactions is crucial for understanding complex biological processes and developing targeted therapies.
Purpose of the Study:
- To develop a scalable platform for massively parallel screening of combinatorial gene perturbations in human cells.
- To translate identified gene pairs into effective drug combinations for modulating human disease phenotypes, specifically ovarian cancer.
Main Methods:
- Utilized CRISPR-Cas9 and Combinatorial Genetics en masse (CombiGEM) to create barcoded combinatorial genetic libraries.
- Performed high-throughput pooled screening of 23,409 dual guide-RNA combinations to identify gene pairs inhibiting ovarian cancer cell growth.
- Validated gene pair effects using CRISPR-Cas knockouts and RNA-interference, and tested synergistic drug pairs.
Main Results:
- Identified specific gene pairs, including epigenetic regulators KDM4C/BRD4 and KDM6B/BRD4, that inhibit ovarian cancer cell growth.
- Demonstrated that drug pairs targeting identified gene pairs exhibit synergistic antiproliferative effects.
- Successfully validated the CombiGEM-CRISPR platform's ability to identify impactful genetic and drug combinations.
Conclusions:
- The CombiGEM-CRISPR platform offers a powerful tool for accelerating the systematic discovery of gene and drug combinations.
- This technology has broad applicability for various biological settings and can advance the development of novel therapeutic strategies for complex diseases.
- The study highlights the potential for translating genetic discoveries into effective combination therapies.
