Biology subjects

Marson, A.

Publications and source records attributed to Marson, A..

9 recordsLinked to original sources

bioRxiv2018

Landscape of stimulation-responsive chromatin across diverse human immune cells

The immune system is controlled by a balanced interplay among specialized cell types transitioning between resting and stimulated states. Despite its importance, the regulatory landscape of this system has not yet been fully characterized. To address this gap, we collected ATAC-seq and RNA-seq data under resting and stimulated conditions for 25 immune cell types from peripheral blood of four healthy individuals, and seven cell types from three fetal thymus samples. We found that stimulation caused widespread chromatin remodeling, including a large class of response elements shared between stimulated B and T cells. Furthermore, several autoimmune traits showed significant heritability in stimulation-responsive elements from distinct cell types, highlighting the critical importance of these cell states in autoimmunity. Use of allele-specific read-mapping identified thousands of variants that alter chromatin accessibility in particular conditions. Notably, variants associated with changes in stimulation-specific chromatin accessibility were not enriched for associations with gene expression regulation in whole blood - a tissue commonly used in eQTL studies. Thus, large-scale maps of variants associated with gene regulation lack a condition important for understanding autoimmunity. As a proof-of-principle we identified variant rs6927172, which links stimulated T cell-specific chromatin dysregulation in the TNFAIP3 locus to ulcerative colitis and rheumatoid arthritis. Overall, our results provide a broad resource of chromatin landscape dynamics and highlight the need for large-scale characterization of effects of genetic variation in stimulated cells.

genomics↗

bioRxiv2018

Systematic characterization of genome editing in primary T cells reveals proximal genomic insertions and enables machine learning prediction of CRISPR-Cas9 DNA repair outcomes

The Streptococcus pyogenes Cas9 (SpCas9) nuclease has become a ubiquitous genome editing tool due to its ability to target almost any location in DNA and create a double-stranded break1,2. After DNA cleavage, the break is fixed with endogenous DNA repair machinery, either by non-templated mechanisms (e.g. non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ)), or homology directed repair (HDR) using a complementary template sequence3,4. Previous work has shown that the distribution of repair outcomes within a cell population is non-random and dependent on the targeted sequence, and only recent efforts have begun to investigate this further5-11. However, no systematic work to date has been validated in primary human cells5,7. Here, we report DNA repair outcomes from 1,521 unique genomic locations edited with SpCas9 ribonucleoprotein complexes (RNPs) in primary human CD4+ T cells isolated from multiple healthy blood donors. We used targeted deep sequencing to measure the frequency distribution of repair outcomes for each guide RNA and discovered distinct features that drive individual repair outcomes after SpCas9 cleavage. Predictive features were combined into a new machine learning model, CRISPR Repair OUTcome (SPROUT), that predicts the length and probability of nucleotide insertions and deletions with R2 greater than 0.5. Surprisingly, we also observed large insertions at more than 90% of targeted loci, albeit at a low frequency. The inserted sequences aligned to diverse regions in the genome, and are enriched for sequences that are physically proximal to the break site due to chromatin interactions. This suggests a new mechanism where sequences from three-dimensionally neighboring regions of the genome can be inserted during DNA repair after Cas9-induced DNA breaks. Together, these findings provide powerful new predictive tools for Cas9-dependent genome editing and reveal new outcomes that can result from genome editing in primary T cells.

cell biology↗

bioRxiv2018

Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function

Human T cells are central effectors of immunity and cancer immunotherapy. CRISPR-based functional studies in T cells could prioritize novel targets for drug development and improve the design of genetically reprogrammed cell-based therapies. However, large-scale CRISPR screens have been challenging in primary human cells. We developed a new method, sgRNA lentiviral infection with Cas9 protein electroporation (SLICE), to identify regulators of stimulation responses in primary human T cells. Genome-wide loss-of-function screens identified essential T cell receptor signaling components and genes that negatively tune proliferation following stimulation. Targeted ablation of individual candidate genes validated hits and identified perturbations that enhanced cancer cell killing. SLICE coupled with single-cell RNA-Seq revealed signature stimulation-response gene programs altered by key genetic perturbations. SLICE genome-wide screening was also adaptable to identify mediators of immunosuppression, revealing genes controlling response to adenosine signaling. The SLICE platform enables unbiased discovery and characterization of functional gene targets in primary cells.

immunology↗

bioRxiv2017

A CRISPR-Cas9 Genome Engineering Platform in Primary CD4+ T Cells for the Interrogation of HIV Host Factors

CRISPR-Cas9 gene editing strategies have revolutionized our ability to engineer the human genome for robust functional interrogation of complex biological processes. We have recently adapted this technology to primary human T cells to generate a high-throughput platform for analyzing the role of host factors in pathogen infection and lifecycle. Here, we describe applications of this system to investigate HIV pathogenesis in CD4+ T cells. Briefly, CRISPR-Cas9 ribonucleoproteins (crRNPs) are synthesized in vitro and delivered to activated primary human CD4+ T cells by nucleofection. These edited cells are then validated and expanded for use in downstream cellular, genetic, or protein-based assays. Our platform supports the arrayed generation of several gene manipulations in only a few hours time and is widely adaptable across culture conditions, infection protocols, and downstream applications. We present detailed protocols for crRNP synthesis, primary T cell culture, 96-well nucleofection, molecular validation, and HIV infection with additional considerations for guide and screen design as well as crRNP multiplexing.

systems biology↗

bioRxiv2017

Light-Activated Cell Identification and Sorting (LACIS): A New Method to Identify and Select Edited Clones on a Microfluidic Device

CRISPR-Cas9 gene editing has revolutionized cell engineering and promises to open new doors in gene and cell therapies. Despite improvements in the CRISPR-editing molecular toolbox in cell lines and primary cells, identifying and purifying properly edited clones remains slow, laborious and low-yield. Here, we establish a new method that uses cell manipulation on a chip with Opto-Electronic Positioning (OEP) technology to enable clonal isolation and selection of edited cells. We focused on editing CXCR4 in primary human T cells, a gene that encodes a co-receptor for HIV entry. T cells hold significant potential for cell-based therapy, but the gene-editing efficiency and expansion potential of these cells is limited. We describe here a method to obviate these limitations. Briefly, after electroporation of cells with CXCR4-targeting Cas9 ribonucleoproteins (RNPs), single T cells were isolated on a chip, where they proliferated over time into well-resolved colonies. Phenotypic consequences of genome editing could be rapidly assessed on-chip with cell-surface staining for CXCR4. Furthermore, independent of phenotype, individual colonies could be identified based on their specific genotype at the 5-10 cell stage. Each colony was split and sequentially exported for immediate on-target sequencing and validation, and further off-chip clonal expansion of the validated clones. We were able to assess single-clone editing efficiencies, including the rate of monoallelic and biallelic indels or precise nucleotide replacements. This new method will enable identification and selection of perfectly edited clones within 10 days from Cas9-RNP introduction in cells based on the phenotype and/or genotype.

bioengineering↗

bioRxiv2017

Correction of autoimmune IL2RA mutations in primary human T cells using non-viral genome targeting

Human T cells are central to physiological immune homeostasis, which protects us from pathogens without collateral autoimmune inflammation. They are also the main effectors in most current cancer immunotherapy strategies1. Several decades of work have aimed to genetically reprogram T cells for therapeutic purposes2-5, but as human T cells are resistant to most standard methods of large DNA insertion these approaches have relied on recombinant viral vectors, which do not target transgenes to specific genomic sites6, 7. In addition, the need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells through homology-directed repair (HDR), but to date in human T cells this still requires viral transduction8, 9. Here, we developed a non-viral, CRISPR-Cas9 genome targeting system that permits the rapid and efficient insertion of individual or multiplexed large (>1 kilobase) DNA sequences at specific sites in the genomes of primary human T cells while preserving cell viability and function. We successfully tested the potential therapeutic use of this approach in two settings. First, we corrected a pathogenic IL2RA mutation in primary T cells from multiple family members with monogenic autoimmune disease and demonstrated enhanced signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR redirecting T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized the tumour antigen, with concomitant cytokine release and tumour cell killing. Taken together, these studies provide preclinical evidence that non-viral genome targeting will enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

genetics↗

bioRxiv2017

Enhancer connectome in primary human cells reveals target genes of disease-associated DNA elements

The challenge of linking intergenic mutations to target genes has limited molecular understanding of diverse human diseases. Here, we show H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in rare primary human T cell subtypes and coronary artery smooth muscle cells. Differentiation of naive T cells to either T helper 17 cells or regulatory T cells create subtype-specific enhancer-promoter interactions, specifically at regions of shared DNA accessibility. These data provide a principled means of assigning molecular functions to autoimmune and cardiovascular disease risk variants, linking hundreds of noncoding variants to putative gene targets. Target genes identified with HiChIP are further supported by CRISPR interference and activation at linked enhancers, by the presence of expression quantitative trait loci, and by allele-specific enhancer loops in patient-derived primary cells. The majority of disease-associated enhancers contact genes beyond the nearest gene in the linear genome, leading to a four-fold increase of potential target genes for autoimmune and cardiovascular diseases.

genomics↗

bioRxiv2017

Multiplexing droplet-based single cell RNA-sequencing using natural genetic barcodes

Droplet-based single-cell RNA-sequencing (dscRNA-seq) has enabled rapid, massively parallel profiling of transcriptomes from tens of thousands of cells. Multiplexing samples for single cell capture and library preparation in dscRNA-seq would enable cost-effective designs of differential expression and genetic studies while avoiding technical batch effects, but its implementation remains challenging. Here, we introduce an in-silico algorithm demuxlet that harnesses natural genetic variation to discover the sample identity of each cell and identify droplets containing two cells. These capabilities enable multiplexed dscRNA-seq experiments where cells from unrelated individuals are pooled and captured at higher throughput than standard workflows. To demonstrate the performance of demuxlet, we sequenced 3 pools of peripheral blood mononuclear cells (PBMCs) from 8 lupus patients. Given genotyping data for each individual, demuxlet correctly recovered the sample identity of > 99% of singlets, and identified doublets at rates consistent with previous estimates. In PBMCs, we demonstrate the utility of multiplexed dscRNA-seq in two applications: characterizing cell type specificity and inter-individual variability of cytokine response from 8 lupus patients and mapping genetic variants associated with cell type specific gene expression from 23 donors. Demuxlet is fast, accurate, scalable and could be extended to other single cell datasets that incorporate natural or synthetic DNA barcodes.

bioinformatics↗

bioRxiv2016

Discovery of an autoimmunity-associated IL2RA enhancer by unbiased targeting of transcriptional activation

The majority of genetic variants associated with common human diseases map to enhancers, non-coding elements that shape cell type-specific transcriptional programs and responses to specific extracellular cues 1-3. In order to understand the mechanisms by which non-coding genetic variation contributes to disease, systematic mapping of functional enhancers and their biological contexts is required. Here, we develop an unbiased discovery platform that can identify enhancers for a target gene without prior knowledge of their native functional context. We used tiled CRISPR activation (CRISPRa) to synthetically recruit transcription factors to sites across large genomic regions (>100 kilobases) surrounding two key autoimmunity risk loci, CD69 and IL2RA (interleukin-2 receptor alpha; CD25). We identified several CRISPRa responsive elements (CaREs) with stimulation-dependent enhancer activity, including an IL2RA enhancer that harbors an autoimmunity risk variant. Using engineered mouse models and genome editing of human primary T cells, we found that sequence perturbation of the disease-associated IL2RA enhancer does not block IL2RA expression, but rather delays the timing of gene activation in response to specific extracellular signals. This work develops an approach to rapidly identify functional enhancers within non-coding regions, decodes a key human autoimmunity association, and suggests a general mechanism by which genetic variation can cause immune dysfunction.

genetics↗