Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM.

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.

Modeling biological copper clusters: synthesis of a tricopper complex, and its chloride- and sulfide-bridged congeners.

The synthesis and characterization of a family of tricopper clusters housed within a tris(ß-diketimine) cyclophane ligand (H3L) that bear structural similarities to biological copper clusters are reported. In all complexes, each Cu atom is held within the N2-chelate of a single ß-diketiminate arm. Reaction of L(3-) with CuCl affords an anionic complex containing a µ3-chloride donor in the central cavity, whereas there is no evidence for bromide incorporation in the product of the reaction of L(3-) with CuBr (Cu3L). Cu3L reacts with elemental sulfur to generate the corresponding air-stable mixed-valent (µ3-sulfido)tricopper complex, Cu3(µ3-S)L, which represents the first example of a sulfide-bridged copper cluster in which each metal center is both coordinatively unsaturated and held within a N-rich environment. The calculated LUMO is predominantly Cu-S π* in character and delocalized over all three metal centers, which is consistent with the isotropic ten-line absorption (g ∼ 2.095, A ∼ 33 G) observed at room temperature in EPR spectra of the one-electron chemically reduced complex, [Cu3(µ3-S)L](-).

Contributions of the S100A9 C-terminal tail to high-affinity Mn(II) chelation by the host-defense protein human calprotectin.

Human calprotectin (CP) is an antimicrobial protein that coordinates Mn(II) with high affinity in a Ca(II)-dependent manner at an unusual histidine-rich site (site 2) formed at the S100A8/S100A9 dimer interface. We present a 16-member CP mutant family where mutations in the S100A9 C-terminal tail (residues 96-114) are employed to evaluate the contributions of this region, which houses three histidines and four acidic residues, to Mn(II) coordination at site 2. The results from analytical size-exclusion chromatography, Mn(II) competition titrations, and electron paramagnetic resonance spectroscopy establish that the C-terminal tail is essential for high-affinity Mn(II) coordination by CP in solution. The studies indicate that His103 and His105 (HXH motif) of the tail complete the Mn(II) coordination sphere in solution, affording an unprecedented biological His6 site. These solution studies are in agreement with a Mn(II)-CP crystal structure reported recently (Damo, S. M.; et al. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 3841). Remarkably high-affinity Mn(II) binding is retained when either H103 or H105 are mutated to Ala, when the HXH motif is shifted from positions 103-105 to 104-106, and when the human tail is substituted by the C-terminal tail of murine S100A9. Nevertheless, antibacterial activity assays employing human CP mutants reveal that the native disposition of His residues is important for conferring growth inhibition against Escherichia coli and Staphylococcus aureus. Within the S100 family, the S100A8/S100A9 heterooligomer is essential for providing high-affinity Mn(II) binding; the S100A7, S100A9(C3S), S100A12, and S100B homodimers do not exhibit such Mn(II)-binding capacity.

Calcium Ions Tune the Zinc-Sequestering Properties and Antimicrobial Activity of Human S100A12.

Human S100A12 is a host-defense protein expressed and released by neutrophils that contributes to innate immunity. Apo S100A12 is a 21-kDa antiparallel homodimer that harbors two Ca(II)-binding EF-hand domains per subunit and exhibits two His3Asp motifs for chelating transition metal ions at the homodimer interface. In this work, we present results from metal-binding studies and microbiology assays designed to ascertain whether Ca(II) ions modulate the Zn(II)-binding properties of S100A12 and further evaluate the antimicrobial properties of this protein. Our metal depletion studies reveal that Ca(II) ions enhance the ability of S100A12 to sequester Zn(II) from microbial growth media. We report that human S100A12 has antifungal activity against Candida albicans, C. krusei, C. glabrata and C. tropicalis, all of which cause human disease. This antifungal activity is Ca(II)-dependent and requires the His3Asp metal-binding sites. We expand upon prior studies of the antibacterial activity of S100A12 and report Ca(II)-dependent and strain-selective behavior. S100A12 exhibited in vitro growth inhibitory activity against Listeria monocytogenes. In contrast, S100A12 had negligible effect on the growth of Escherichia coli K-12 and Pseudomonas aeruginosa PAO1. Loss of functional ZnuABC, a high-affinity Zn(II) import system, increased the susceptibility of E. coli and P. aeruginosa to S100A12, indicating that S100A12 deprives these mutant strains of Zn(II). To evaluate the Zn(II)-binding sites of S100A12 in solution, we present studies using Co(II) as a spectroscopic probe and chromophoric small-molecule chelators in Zn(II) competition titrations. We confirm that S100A12 binds Zn(II) with a 2:1 stoichiometry, and our data indicate sub-nanomolar affinity binding. Taken together, these data support a model whereby S100A12 uses Ca(II) ions to tune its Zn(II)-chelating properties and antimicrobial activity.

Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq.

To explore the distinct genotypic and phenotypic states of melanoma tumors, we applied single-cell RNA sequencing (RNA-seq) to 4645 single cells isolated from 19 patients, profiling malignant, immune, stromal, and endothelial cells. Malignant cells within the same tumor displayed transcriptional heterogeneity associated with the cell cycle, spatial context, and a drug-resistance program. In particular, all tumors harbored malignant cells from two distinct transcriptional cell states, such that tumors characterized by high levels of the MITF transcription factor also contained cells with low MITF and elevated levels of the AXL kinase. Single-cell analyses suggested distinct tumor microenvironmental patterns, including cell-to-cell interactions. Analysis of tumor-infiltrating T cells revealed exhaustion programs, their connection to T cell activation and clonal expansion, and their variability across patients. Overall, we begin to unravel the cellular ecosystem of tumors and how single-cell genomics offers insights with implications for both targeted and immune therapies.