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.

Keywords: CRISPR-Cas; CombiGEM; genetic perturbations; high-throughput screening; multifactorial genetics.

Fig. 1.

Strategy for assembling barcoded combinatorial gRNA libraries. Barcoded gRNA oligo pairs were synthesized, annealed, and cloned in storage vectors in pooled format. Oligos with the gRNA scaffold sequence were inserted into the pooled storage vector library to create the barcoded sgRNA library. Detailed assembly steps are described in SI Appendix, Fig. S1. The CombiGEM strategy was used to build the combinatorial gRNA library. Pooled barcoded sgRNA inserts prepared from the sgRNA library with BglII and MfeI digestion were ligated via compatible overhangs generated in the destination vectors with BamHI and EcoRI digestion. Iterative one-pot ligation created (n)-wise gRNA libraries with unique barcodes corresponding to the gRNAs concatenated at one end, thus enabling tracking of individual combinatorial members within pooled populations via next-generation sequencing.

Fig. 2.

High-throughput screen identifies gRNA combinations that inhibit cancer cell proliferation. (A) OVCAR8-ADR-Cas9 cells infected with the barcoded two-wise gRNA library were cultured for 15 and 20 d. Barcode representations within the cell pools were quantified using Illumina HiSeq. (B) Two-wise gRNA combinations that modulated proliferation were ranked by log₂ ratios between their normalized barcode counts in 20-d versus 15-d cultured cells (Right). To enable comparisons between the two-wise gRNA combinations with their sgRNA counterparts, the same data for sgRNAs paired with control sgRNAs (Left) are also plotted. Combinations with control gRNA pairs are highlighted in orange. The antiproliferative effects of gRNA combinations that were confirmed in another biological replicate are highlighted in blue (SI Appendix, Fig. S9). The labeled gRNA combinations were further validated in this study. (C) Individual validation of two-wise combinations that modulated cancer cell growth. OVCAR8-ADR-Cas9 cell populations individually infected with lentiviruses expressing the indicated two-wise gRNA combinations were cultured for 15 d, and an equal number of cells was then replated and cultured for additional time periods as indicated. Cell viability was measured by the MTT assay, and characterized by absorbance measurements (OD₅₇₀–OD₆₅₀) (n = 3). Data represent mean ± SD.

Fig. 3.

Combinatorial inhibition of KDM4C and BRD4, as well as KDM6B and BRD4, inhibits human ovarian cancer cell growth. (A and B) OVCAR8-ADR-Cas9 cells infected with lentiviruses expressing the indicated single or combinatorial gRNAs (A) or OVCAR8-ADR cells coinfected with lentiviruses expressing the indicated shRNAs (B) were cultured for 15 d and 9 d, respectively. Equal numbers of infected cells were then replated and cultured for an additional 5 d (A) and 4 d (B). Cell viabilities relative to control sgRNA (A) or shRNA (B) were determined by the MTT assay. (C and D) OVCAR8-ADR cells were treated with the indicated drugs for 5 d (C) and 7 d (D). JQ1 (36), SD70 (35), and GSK-J4 (37) are small-molecule inhibitors of BRD4, KDM4C, and KDM6B/6A, respectively. Percentage inhibition of cell growth relative to no drug control was determined by the MTT assay. The calculated excess inhibition over the predicted Bliss independence and HSA models was shown for each drug combination pair. Data represent mean ± SD (n = 3 for A; n = 6 for B–D) from biological replicates. *P < 0.05 and ^#P < 0.05 represent significant differences between the indicated samples and between drug-treated versus no drug control samples, respectively.