Pamela Milani

Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM

Significance The systematic discovery of new gene and drug combinations that modulate complex biological phenotypes and human diseases requires scalable and multiplexed screening technologies. We leverage the programmability of the CRISPR-Cas9 system for multiplexed targeting of specific genomic loci and the versatility of the combinatorial genetics en masse (CombiGEM) technology to rapidly assemble barcoded combinatorial genetic perturbation libraries that can be tracked with high-throughput sequencing. CombiGEM-CRISPR enables simple, massively parallel screening of barcoded combinatorial gene perturbations in human cells, and the translation of these hits into effective drug combinations. This approach is broadly applicable for performing pooled combinatorial genetic perturbations to map out how the orchestrated action of genes controls complex phenotypes and to translate these findings into novel drug combinations. 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.

Cell freezing protocol suitable for ATAC-Seq on motor neurons derived from human induced pluripotent stem cells.

In recent years, the assay for transposase-accessible chromatin using sequencing (ATAC-Seq) has become a fundamental tool of epigenomic research. However, it is difficult to perform this technique on frozen samples because freezing cells before extracting nuclei can impair nuclear integrity and alter chromatin structure, especially in fragile cells such as neurons. Our aim was to develop a protocol for freezing neuronal cells that is compatible with ATAC-Seq; we focused on a disease-relevant cell type, namely motor neurons differentiated from induced pluripotent stem cells (iMNs) from a patient affected by spinal muscular atrophy. We found that while flash-frozen iMNs are not suitable for ATAC-Seq, the assay is successful with slow-cooled cryopreserved cells. Using this method, we were able to isolate high quality, intact nuclei, and we verified that epigenetic results from fresh and cryopreserved iMNs quantitatively agree.

Network Modeling Identifies Patient-specific Pathways in Glioblastoma.

Glioblastoma is the most aggressive type of malignant human brain tumor. Molecular profiling experiments have revealed that these tumors are extremely heterogeneous. This heterogeneity is one of the principal challenges for developing targeted therapies. We hypothesize that despite the diverse molecular profiles, it might still be possible to identify common signaling changes that could be targeted in some or all tumors. Using a network modeling approach, we reconstruct the altered signaling pathways from tumor-specific phosphoproteomic data and known protein-protein interactions. We then develop a network-based strategy for identifying tumor specific proteins and pathways that were predicted by the models but not directly observed in the experiments. Among these hidden targets, we show that the ERK activator kinase1 (MEK1) displays increased phosphorylation in all tumors. By contrast, protein numb homolog (NUMB) is present only in the subset of the tumors that are the most invasive. Additionally, increased S100A4 is associated with only one of the tumors. Overall, our results demonstrate that despite the heterogeneity of the proteomic data, network models can identify common or tumor specific pathway-level changes. These results represent an important proof of principle that can improve the target selection process for tumor specific treatments.

Chromatin Landscape and Epigenetic Signatures in Neurological Disorders: Emerging Perspectives for Biological and Clinical Research

The chapter provides an overview of the recent breakthroughs in the understanding of the role of epigenetic mechanisms, such as histone posttranslational modifications and DNA methylation, in the onset and progression of two neurodegenerative disorders, Huntingtons disease and amyotrophic lateral sclerosis. We also summarize the latest advances in the development of safe and effective therapeutic approaches based on the modulation of epigenetic mechanisms in these disorders. In particular, preclinical investigation proved the ability of several histone deacetylase inhibitors and compounds targeting DNA methylation to slow disease progression and improve clinical parameters. Elucidating the epigenetic components of neurodegeneration may also lead to the identification of disease-specific signatures, paving the way for the discovery of new biological markers.