The Library of Integrated Network-Based Cellular Signatures (LINCS) is an NIH Common Fund program that catalogs how human cells globally respond to chemical, genetic, and disease perturbations. Resources generated by LINCS include experimental and computational methods, visualization tools, molecular and imaging data, and signatures. By assembling an integrated picture of the range of responses of human cells exposed to many perturbations, the LINCS program aims to better understand human disease and to advance the development of new therapies. Perturbations under study include drugs, genetic perturbations, tissue micro-environments, antibodies, and disease-causing mutations. Responses to perturbations are measured by transcript profiling, mass spectrometry, cell imaging, and biochemical methods, among other assays. The LINCS program focuses on cellular physiology shared among tissues and cell types relevant to an array of diseases, including cancer, heart disease, and neurodegenerative disorders. This Perspective describes LINCS technologies, datasets, tools, and approaches to data accessibility and reusability.

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

Answer ALS is a biological and clinical resource of patient-derived, induced pluripotent stem (iPS) cell lines, multi-omic data derived from iPS neurons and longitudinal clinical and smartphone data from over 1,000 patients with ALS. This resource provides population-level biological and clinical data that may be employed to identify clinical–molecular–biochemical subtypes of amyotrophic lateral sclerosis (ALS). A unique smartphone-based system was employed to collect deep clinical data, including fine motor activity, speech, breathing and linguistics/cognition. The iPS spinal neurons were blood derived from each patient and these cells underwent multi-omic analytics including whole-genome sequencing, RNA transcriptomics, ATAC-sequencing and proteomics. The intent of these data is for the generation of integrated clinical and biological signatures using bioinformatics, statistics and computational biology to establish patterns that may lead to a better understanding of the underlying mechanisms of disease, including subgroup identification. A web portal for open-source sharing of all data was developed for widespread community-based data analytics.

Aberrant neuronal development and the persistence of mitotic cellular populations have been implicated in a multitude of neurological disorders, including Huntington's disease (HD). However, the mechanism underlying this potential pathology remains unclear. We used a modified protocol to differentiate induced pluripotent stem cells (iPSCs) from HD patients and unaffected controls into neuronal cultures enriched for medium spiny neurons, the cell type most affected in HD. We performed single-cell and bulk transcriptomic and epigenomic analyses and demonstrated that a persistent cyclin D1 + neural stem cell (NSC) population is observed selectively in adult-onset HD iPSCs during differentiation. Treatment with a WNT inhibitor abrogates this NSC population while preserving neurons. Taken together, our findings identify a mechanism that may promote aberrant neurodevelopment and adult neurogenesis in adult-onset HD striatal neurons with the potential for therapeutic compensation.

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.Since its establishment, the assay for transposase-accessible chromatin using sequencing (ATAC-Seq) has revolutionized the study of epigenetics 1,2 . This technique detects open-chromatin regions and maps transcription factor binding events genome-wide by means of direct in vitro transposition of native chromatin. Specifically, hyperactive Tn5 transposase is used to interrogate chromatin accessibility by inserting high-throughput DNA sequencing adapters into open genomic regions, which allows for the preferential amplification of DNA fragments located at sites of active chromatin. Because the DNA sites directly bound by DNA-binding proteins are protected from transposition, this approach enables the inference of transcription factor occupancy at the level of individual functional regulatory regions. Furthermore, ATAC-Seq can be utilized to decode nucleosome occupancy and positioning, by exploiting the fact that the Tn5 transposase cuts DNA with a periodicity of about 150-200 bp, corresponding to the length of the DNA fragments wrapped around histones 3 . This periodicity is maintained up to six nucleosomes and provides information about the spatial organization of nucleosomes within accessible chromatin. ATAC-Seq signals thus allow for the delineation of fine-scale architectures of the regulatory framework by correlating occupancy patterns with other features, such as chromatin remodeling and global gene induction programs. Compared to other epigenetic methodologies, such as FAIRE-Seq and conventional DNase-Seq, ATAC-Seq requires a small number of cells. Therefore, it is suitable for work on precious samples, including differentiated cells derived from induced pluripotent stem cells (iPSCs), primary cell culture, and limited clinical specimens. Recently developed techniques, such as single-cell DNase sequencing (scDNase-seq) 4 , indexing-first ChIP-Seq (iChIP) 5 , ultra-low-input micrococcal nuclease-based native ChIP (ULI-NChIP) 6 , and ChIPmentation 7 , allow for the epigenomic investigation of small number of cells or even single cells without requiring microfluidic devices. However, these ...