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 (Combi-GEM) 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

Cancer Drug Development Using Drosophila as an in vivo Tool: From Bedside to Bench and Back

“…The technology has been used to identify genes required for the development of drug resistance in cancer [102,103]. The tool has also been used to uncover the mechanism of action, resistance [104], and efficacy of drug combinations [105] in human cancer cells. However, the tool has yet to be employed in Drosophila cancer research.…”

High-Throughput Approaches to Pinpoint Function within the Noncoding Genome

“…The majority of pooled CRISPR screens have used the Cas9 nuclease for gene knockout and have been performed at genome-wide scale (Koike-Yusa et al, 2014; Shalem et al, 2014, 2015; Wang et al, 2014). Other gene-targeting strategies have included paired gRNAs to target two genes in a single cell (Han et al, 2017; Shen et al, 2017; Wong et al, 2016), mutagenesis to identify gain-of-function mutations resulting from in-frame NHEJ repair (Donovan et al, 2017), base editing to introduce stop codons to knock out genes (Kuscu et al, 2017), protein domain targeting (Shi et al, 2015), and in vivo screening for phenotypic selection not possible to perform in vitro (Chen et al, 2015; Xu et al, 2017). In addition to mutagenesis with Cas9 nuclease, genome-wide screens have also been performed with CRISPRa/i systems for gene activation/repression (Gilbert et al, 2014; Konermann et al, 2015).…”

Mammalian synthetic biology in the age of genome editing and personalized medicine

“…Construction of multiplexed, barcoded CRISPR/Cas9 sgRNA libraries has rapidly evolved to allow combinatorial perturbations and the discovery of cooperative epigenetic modifications that play a role in oncogenesis [36]. Stockman et al developed a multiplex strategy (MoSAIC) for assessing genetic interactions using CRISPR/Cas9, in which direct barcoding and pairwise assembly of sgRNAs occur in a single cloning step, further improving assembly efficiency and reducing potential library bias associated with multi-step construction methods [37].…”