Biology subjects

Hess, G. T.

Publications and source records attributed to Hess, G. T..

10 recordsLinked to original sources

bioRxiv2024

Towards optimizing diversifying base editors for high-throughput studies of single-nucleotide variants

Determining the phenotypic effects of single nucleotide variants is critical for understanding the genome and interpreting clinical sequencing results. Base editors, including diversifying base editors that create C>N mutations, are potent tools for installing point mutations in mammalian genomes and studying their effect on cellular function. Numerous base editor options are available for such studies, but little information exists on how the composition of the editor (deaminase, recruitment method, and fusion architecture) affects editing. To address this knowledge gap, the effect of various design features, such as deaminase recruitment and delivery method (electroporation or lentiviral transduction), on editing was assessed across [~]200 synthetic target sites. The direct fusion of a hyperactive variant of activation-induced cytidine deaminase to the N-terminus of dCas9 (DivA-BE) produced the highest editing efficiency, [~]4-fold better than the previous CRISPR-X method. Additionally, DivA-BE mutagenized the DNA strand that anneals to the targeting sgRNA to create G>N mutations, which were absent when the deaminase was fused to the C-terminus of dCas9. The DivA-BE editors efficiently diversified their target sites, an ideal characteristic for discovering functional variants. These and other findings provide a comprehensive analysis of how design features influence the activity of several popular base editors.

genomics↗

bioRxiv2023

Development of compact transcriptional effectors using high-throughput measurements in diverse contexts

Human nuclear proteins contain >1000 transcriptional effector domains that can activate or repress transcription of target genes. We lack a systematic understanding of which effector domains regulate transcription robustly across genomic, cell-type, and DNA-binding domain (DBD) contexts. Here, we developed dCas9-mediated high-throughput recruitment (HT-recruit), a pooled screening method for quantifying effector function at endogenous targets, and tested effector function for a library containing 5092 nuclear protein Pfam domains across varied contexts. We find many effectors depend on target and DBD contexts, such as HLH domains that can act as either activators or repressors. We then confirm these findings and further map context dependencies of effectors drawn from unannotated protein regions using a larger library containing 114,288 sequences tiling chromatin regulators and transcription factors. To enable efficient perturbations, we select effectors that are potent in diverse contexts, and engineer (1) improved ZNF705 KRAB CRISPRi tools to silence promoters and enhancers, and (2) a compact human activator combination NFZ for better CRISPRa and inducible circuit delivery. Together, this effector-by-context functional map reveals context-dependence across human effectors and guides effector selection for robustly manipulating transcription.

systems biology↗

bioRxiv2022

Identification of orphan ligand-receptor relationships using a cell-based CRISPRa enrichment screening platform.

Secreted proteins, which include cytokines, hormones and growth factors, are extracellular ligands that control key signaling pathways mediating cell-cell communication within and between tissues and organs. Many drugs target secreted ligands and their cell-surface receptors. Still, there are hundreds of secreted human proteins that either have no identified receptors ("orphans") and are likely to act through cell surface receptors that have not yet been characterized. Discovery of secreted ligand-receptor interactions by high-throughput screening has been problematic, because the most commonly used high-throughput methods for protein-protein interaction (PPI) screening do not work well for extracellular interactions. Cell-based screening is a promising technology for definition of new ligand-receptor interactions, because multimerized ligands can enrich for cells expressing low affinity cell-surface receptors, and such methods do not require purification of receptor extracellular domains. Here, we present a proteo-genomic cell-based CRISPR activation (CRISPRa) enrichment screening platform employing customized pooled cell surface receptor sgRNA libraries in combination with a magnetic bead selection-based enrichment workflow for rapid, parallel ligand-receptor deorphanization. We curated 80 potentially high value orphan secreted proteins and ultimately screened 20 secreted ligands against two cell sgRNA libraries with targeted expression of all single-pass (TM1) or multi-pass (TM2+) receptors by CRISPRa. We identified previously unknown interactions in 12 of these screens, and validated several of them using surface plasmon resonance and/or cell binding. The newly deorphanized ligands include three receptor tyrosine phosphatase (RPTP) ligands and a chemokine like protein that binds to killer cell inhibitory receptors (KIRs). These new interactions provide a resource for future investigations of interactions between the human secreted and membrane proteomes.

cell biology↗

bioRxiv2020

High-throughput discovery and characterization of human transcriptional effectors

Thousands of proteins localize to the nucleus; however, it remains unclear which contain transcriptional effectors. Here, we develop HT-recruit - a pooled assay where protein libraries are recruited to a reporter, and their transcriptional effects are measured by sequencing. Using this approach, we measure gene silencing and activation for thousands of domains. We find a relationship between repressor function and evolutionary age for the KRAB domains, discover Homeodomain repressor strength is collinear with Hox genetic organization, and identify activities for several Domains of Unknown Function. Deep mutational scanning of the CRISPRi KRAB maps the co-repressor binding surface and identifies substitutions that improve stability/silencing. By tiling 238 proteins, we find repressors as short as 10 amino acids. Finally, we report new activator domains, including a divergent KRAB. Together, these results provide a resource of 600 human proteins containing effectors and demonstrate a scalable strategy for assigning functions to protein domains.

genetics↗

bioRxiv2020

Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation

Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe GalNAzMe that is specific for cancer-relevant Ser/Thr-N-acetylgalactosamine (O-GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn (N)-linked glycans. We equip cells with the capacity to biosynthesize the nucleotide-sugar donor UDP-GalNAzMe from a caged precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR knock-out (KO) screen, and imaging of intestinal organoids. GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation. Significance statementA large portion of all secreted and cell surface proteins in humans are modified by Ser/Thr(O)-linked glycosylation with N-acetylgalactosamine (GalNAc). While of fundamental importance in health and disease, O-GalNAc glycosylation is technically challenging to study because of a lack of specific tools to be used in biological assays. Here, we design an O-GalNAc specific reporter molecule termed GalNAzMe to selectively label O-GalNAc glycoproteins in living human cells. GalNAzMe is compatible with a range of experiments in quantitative biology to broaden our understanding of glycosylation. We further demonstrate that labeling is genetically programmable by expression of a mutant glycosyltransferase, allowing application even to experiments with low inherent sensitivity.

synthetic biology↗

bioRxiv2020

The LRRC8A:C Heteromeric Channel Is a cGAMP Transporter and the Dominant cGAMP Importer in Human Vasculature Cells

Extracellular 23-cyclic-GMP-AMP (cGAMP) is an immunotransmitter secreted by cancer cells and taken up by host cells to activate the anti-cancer STING pathway. No cGAMP exporter has been identified, and SLC19A1, a recently identified cGAMP importer, does not account for the import activity in most cell types. Here, we identify the LRRC8A:C heteromeric channel, a volume-regulated anion channel (VRAC), as a cGAMP transporter. This channel mediates cGAMP import or export depending on the cGAMP chemical gradient, and channel activation or inhibition modulates cGAMP transport. Other 23-cyclic dinucleotides are also transported by LRRC8A:C channels, including the investigational cancer therapeutic ADU-S100. Furthermore, we demonstrate that the LRRC8A-containing channel is the dominant cGAMP importer in primary human vasculature cells. Given tumor vasculatures regulation of immune infiltration and its disruption in response to STING agonists, we have uncovered a leading molecular mechanism for extracellular cGAMP signaling in this important anti-cancer target.

biochemistry↗

bioRxiv2019

SLC19A1 is an importer of the immunotransmitter cGAMP

23-cyclic-GMP-AMP (cGAMP) is a second messenger that activates the antiviral Stimulator of Interferon Genes (STING) pathway. We recently identified a novel role for cGAMP as a soluble, extracellular immunotransmitter that is produced and secreted by cancer cells. Secreted cGAMP is then sensed by host cells, eliciting an antitumoral immune response. Due to the antitumoral effects of cGAMP, other CDN-based STING agonists are currently under investigation in clinical trials for metastatic solid tumors. However, it is unknown how cGAMP and other CDNs cross the cell membrane to activate intracellular STING. Using a genome-wide CRISPR screen we identified SLC19A1 as the first known importer of cGAMP and other CDNs, including the investigational new drug 2'3'-bisphosphosphothioate-cyclic-di-AMP (2'3'-CDAS). These discoveries will provide insight into cGAMPs role as an immunotransmitter and aid in the development of more targeted CDN-based cancer therapeutics.

biochemistry↗

bioRxiv2019

Identification and mitigation of pervasive off-target activity in CRISPR-Cas9 screens for essential non-coding elements

Pooled CRISPR-Cas9 screens have recently emerged as a powerful method for functionally characterizing regulatory elements in the non-coding genome, but off-target effects in these experiments have not been systematically evaluated. Here, we conducted a genome-scale screen for essential CTCF loop anchors in the K562 leukemia cell line. Surprisingly, the primary drivers of signal in this screen were single guide RNAs (sgRNAs) with low specificity scores. After removing these guides, we found that there were no CTCF loop anchors critical for cell growth. We also observed this effect in an independent screen fine-mapping the core motifs in enhancers of the GATA1 gene. We then conducted screens in parallel with CRISPRi and CRISPRa, which do not induce DNA damage, and found that an unexpected and distinct set of off-targets also caused strong confounding growth effects with these epigenome-editing platforms. Promisingly, strict filtering of CRISPRi libraries using GuideScan specificity scores removed these confounded sgRNAs and allowed for the identification of essential enhancers, which we validated extensively. Together, our results show off-target activity can severely limit identification of essential functional motifs by active Cas9, while strictly filtered CRISPRi screens can be reliably used for assaying larger regulatory elements.

genomics↗

bioRxiv2017

A comprehensive portrait of cilia and ciliopathies from a CRISPR-based screen for Hedgehog signaling

The primary cilium organizes Hedgehog signaling, shapes embryonic development and is the unifying cause of the ciliopathies. We conducted a functional genomic screen for Hedgehog signaling by engineering antibiotic-based selection of Hedgehog-responsive cells and applying genome-wide CRISPR-mediated gene disruption. The screen robustly identifies factors required for ciliary signaling with few false positives or false negatives. Characterization of hit genes uncovers novel components of several ciliary structures including a protein complex containing {varepsilon}- and {delta}- tubulin that is required for centriole maintenance. The screen also provides an unbiased tool for classifying ciliopathies and reveals that many forms of congenital heart defects are ciliopathies. Collectively, this screen enables a systematic analysis of ciliary function and of ciliopathies and also defines a versatile platform for dissecting signaling pathways through CRISPR-based screening.

genetics↗

bioRxiv2017

Static And Dynamic DNA Loops Form AP-1 Bound Activation Hubs During Macrophage Development

The three-dimensional arrangement of the human genome comprises a complex network of structural and regulatory chromatin loops important for coordinating changes in transcription during human development. To better understand the mechanisms underlying context-specific 3D chromatin structure and transcription during cellular differentiation, we generated comprehensive in situ Hi-C maps of DNA loops during human monocyte-to-macrophage differentiation. We demonstrate that dynamic looping events are regulatory rather than structural in nature and uncover widespread coordination of dynamic enhancer activity at preformed and acquired DNA loops. Enhancer-bound loop formation and enhancer-activation of preformed loops represent two distinct modes of regulation that together form multi-loop activation hubs at key macrophage genes. Activation hubs connect 3.4 enhancers per promoter and exhibit a strong enrichment for Activator Protein 1 (AP-1) binding events, suggesting multi-loop activation hubs driven by cell-type specific transcription factors may represent an important class of regulatory chromatin structures for the spatiotemporal control of transcription.\n\nHIGHLIGHTSO_LIHigh resolution and high sensitivity of loop detection via deeply sequenced in situ Hi-C experiments during monocyte to macrophage differentiation (> 10 billion total reads)\nC_LIO_LIMulti-loop interaction communities identified surrounding key macrophage genes.\nC_LIO_LIMulti-loop communities connect dynamic enhancers through both static and newly acquired DNA loops, forming hubs of activation\nC_LIO_LIMacrophage activation hubs are enriched for AP-1 bound long-range enhancer interactions, suggesting cell-type specific TFs drive changes in 3D structure and transcription through regulatory DNA loops\nC_LI

genomics↗