Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM.

Proc Natl Acad Sci U S A
Authors
Keywords
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.

Year of Publication
2016
Journal
Proc Natl Acad Sci U S A
Volume
113
Issue
9
Pages
2544-9
Date Published
2016 Mar 01
ISSN
1091-6490
URL
DOI
10.1073/pnas.1517883113
PubMed ID
26864203
PubMed Central ID
PMC4780610
Links
Grant list
R01NS089076 / NS / NINDS NIH HHS / United States
P30 CA014051 / CA / NCI NIH HHS / United States
P50GM098792 / GM / NIGMS NIH HHS / United States
P50 GM098792 / GM / NIGMS NIH HHS / United States
DP2OD008435 / OD / NIH HHS / United States
DP2 OD008435 / OD / NIH HHS / United States
R01 NS089076 / NS / NINDS NIH HHS / United States