Biology subjects

Parker, K. R.

Publications and source records attributed to Parker, K. R..

9 recordsLinked to original sources

bioRxiv2023

Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells

CRISPR-Cas9 genome editing has enabled advanced T cell therapies, but occasional loss of the targeted chromosome remains a safety concern. To investigate whether Cas9-induced chromosome loss is a universal phenomenon and evaluate its clinical significance, we conducted a systematic analysis in primary human T cells. Arrayed and pooled CRISPR screens revealed that chromosome loss was generalizable across the genome and resulted in partial and entire loss of the chromosome, including in pre-clinical chimeric antigen receptor T cells. T cells with chromosome loss persisted for weeks in culture, implying the potential to interfere with clinical use. A modified cell manufacturing process, employed in our first-in-human clinical trial of Cas9-engineered T cells,1 dramatically reduced chromosome loss while largely preserving genome editing efficacy. Expression of p53 correlated with protection from chromosome loss observed in this protocol, suggesting both a mechanism and strategy for T cell engineering that mitigates this genotoxicity in the clinic.

cell biology↗

bioRxiv2022

Bidirectional epigenetic editing reveals hierarchies in gene regulation

CRISPR perturbations are valuable tools for studying the functional effects of the genome. However, existing methods are limited in their utility for studying noncoding elements and genetic interactions. Here, we develop a system for bidirectional epigenetic editing (CRISPRai), in which orthogonal activating (CRISPRa) and repressive (CRISPRi) perturbations are applied simultaneously to multiple loci the same cell. We developed dual-gRNA-capture single-cell Perturb-seq to study the established interaction between SPI1 and GATA1, two hemopoietic lineage transcription factors, and discovered novel context-specific regulation modes for co-regulated genes. Extending CRISPRai to noncoding elements, we addressed how multiple enhancers interact to modulate expression of a shared target gene, Interleukin-2, in T cells. We found that enhancer function was primarily additive and enabled fine-tuning of gene expression, yet a clear hierarchy existed among enhancers in strength of gene expression control. The promoter was dominant over most enhancers in controlling gene expression; however, a small subset of enhancers exhibited strong functional effects, or gatekeeper function, and could turn off the gene despite promoter activation. Integration of these functional data with histone ChIP-seq and TF motif enrichment suggests the existence of multiple modes of enhancer-mediated gene regulation. Our method, CRISPRai for bidirectional epigenetic editing, provides an approach for identifying novel genetic interactions that may be overlooked when studied without bidirectional perturbations and can be applied to both genes and noncoding elements.

genomics↗

bioRxiv2021

Systematic discovery of receptor-ligand biology by engineered cell entry and single-cell genomics

Cells communicate with each other via receptor-ligand interactions on the cell surface. Here we describe a technology for lentiviral-mediated cell entry by engineered receptor-ligand interaction (ENTER) to decode receptor specificity. Engineered lentiviral particles displaying specific ligands deliver fluorescent proteins into target cells upon cognate receptor-ligand interaction, without genome integration or transgene transcription. We optimize ENTER to decode interactions between T cell receptor (TCR)-MHC peptides, antibody-antigen, and other receptor-ligand pairs. We develop an effective presentation strategy to capture interactions between B cell receptor (BCR) and intracellular antigen epitopes. Single-cell readout of ENTER by RNA sequencing (ENTER-seq) enables multiplexed enumeration of TCR-antigen specificities, clonality, cell type, and cell states of individual T cells. ENTER-seq of patient blood samples after CMV infection reveals the viral epitopes that drive human effector memory T cell differentiation and inter-clonal phenotypic diversity that targets the same epitope. ENTER enables systematic discovery of receptor specificity, linkage to cell fates, and cell-specific delivery of gene or protein payloads. HIGHLIGHTSO_LIENTER displays ligands, deliver cargos, and records receptor specificity. C_LIO_LIENTER deorphanizes antigen recognition of TCR and BCR. C_LIO_LIENTER-seq maps TCR specificity, clonality and cell state in single cells. C_LIO_LIENTER-seq of patient sample decodes antiviral T cell memory. C_LI

genomics↗

bioRxiv2021

RNA-BINDING PROTEINS DIRECT MYOGENIC CELL FATE DECISIONS

RNA-binding proteins (RBPs) are essential for skeletal muscle regeneration and RBP dysfunction causes muscle degeneration and neuromuscular disease. How ubiquitously expressed RBPs orchestrate complex tissue regeneration and direct cell fate decisions in skeletal muscle remains poorly understood. Single cell RNA-sequencing of regenerating skeletal muscle reveals that RBP expression, including numerous neuromuscular disease-associated RBPs, is temporally regulated in skeletal muscle stem cells and correlates to stages of myogenic differentiation. By combining machine learning with RBP engagement scoring, we discover that the neuromuscular disease associated RBP Hnrnpa2b1 is a differentiation-specifying regulator of myogenesis controlling myogenic cell fate transitions during terminal differentiation. The timing of RBP expression specifies cell fate transitions by providing a layer of post-transcriptional regulation needed to coordinate stem cell fate decisions during complex tissue regeneration.

molecular biology↗

bioRxiv2020

Systematic discovery and functional interrogation of SARS-CoV-2 viral RNA-host protein interactions during infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of a pandemic with growing global mortality. There is an urgent need to understand the molecular pathways required for host infection and anti-viral immunity. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with viral ChIRP-MS data from three other positive-sense RNA viruses defined pan-viral and SARS-CoV-2-specific host interactions. Functional interrogation of these factors with a genome-wide CRISPR screen revealed that the vast majority of viral RNA-binding proteins protect the host from virus-induced cell death, and we identified known and novel anti-viral proteins that regulate SARS-CoV-2 pathogenicity. Finally, our RNA-centric approach demonstrated a physical connection between SARS-CoV-2 RNA and host mitochondria, which we validated with functional and electron microscopy data, providing new insights into a more general virus-specific protein logic for mitochondrial interactions. Altogether, these data provide a comprehensive catalogue of SARS-CoV-2 RNA-host protein interactions, which may inform future studies to understand the mechanisms of viral pathogenesis, as well as nominate host pathways that could be targeted for therapeutic benefit. Highlights{middle dot} ChIRP-MS of SARS-CoV-2 RNA identifies a comprehensive viral RNA-host protein interaction network during infection across two species {middle dot} Comparison to RNA-protein interaction networks with Zika virus, dengue virus, and rhinovirus identify SARS-CoV-2-specific and pan-viral RNA protein complexes and highlights distinct intracellular trafficking pathways {middle dot} Intersection of ChIRP-MS and genome-wide CRISPR screens identify novel SARS-CoV-2-binding proteins with pro- and anti-viral function {middle dot} Viral RNA-RNA and RNA-protein interactions reveal specific SARS-CoV-2-mediated mitochondrial dysfunction during infection

microbiology↗

bioRxiv2020

Transient "rest" induces functional reinvigoration and epigenetic remodeling in exhausted CAR-T cells

T cell exhaustion limits immune responses against cancer and is a major cause of resistance to CAR-T cell therapeutics. Using a model wherein tonic CAR signaling induces hallmark features of exhaustion, we employed a drug-regulatable CAR to test the impact of transient cessation of receptor signaling (i.e. "rest") on the development and maintenance of exhaustion. Induction of rest in exhausting or already-exhausted CAR-T cells resulted in acquisition of a memory-like phenotype, improved anti-tumor functionality, and wholescale transcriptional and epigenetic reprogramming. Similar results were achieved with the Src kinase inhibitor dasatinib, which reversibly suppresses CAR signaling. The degree of functional reinvigoration was proportional to the duration of rest and was associated with expression of transcription factors TCF1 and LEF1. This work demonstrates that transient cessation of CAR-T cell signaling can enhance anti-tumor potency by preventing or reversing exhaustion and challenges the paradigm that exhaustion is an epigenetically fixed state.

immunology↗

bioRxiv2018

Atlas of Subcellular RNA Localization Revealed by APEX-seq

We introduce APEX-seq, a method for RNA sequencing based on spatial proximity to the peroxidase enzyme APEX2. APEX-seq in nine distinct subcellular locales produced a nanometer-resolution spatial map of the human transcriptome, revealing extensive and exquisite patterns of localization for diverse RNA classes and transcript isoforms. We uncover a radial organization of the nuclear transcriptome, which is gated at the inner surface of the nuclear pore for cytoplasmic export of processed transcripts. We identify two distinct pathways of messenger RNA localization to mitochondria, each associated with specific sets of transcripts for building complementary macromolecular machines within the organelle. APEX-seq should be widely applicable to many systems, enabling comprehensive investigations of the spatial transcriptome.

cell biology↗

bioRxiv2018

Coupled single-cell CRISPR screening and epigenomic profiling reveals causal gene regulatory networks

Here we present Perturb-ATAC, a method which combines multiplexed CRISPR interference or knockout with genome-wide chromatin accessibility profiling in single cells, based on the simultaneous detection of CRISPR guide RNAs and open chromatin sites by assay of transposase-accessible chromatin with sequencing (ATAC-seq). We applied Perturb-ATAC to transcription factors (TFs), chromatin-modifying factors, and noncoding RNAs (ncRNAs) in [~]4,300 single cells, encompassing more than 63 unique genotype-phenotype relationships. Perturb-ATAC in human B lymphocytes uncovered regulators of chromatin accessibility, TF occupancy, and nucleosome positioning, and identified a hierarchical organization of TFs that govern B cell state, variation, and disease-associated cis-regulatory elements. Perturb-ATAC in primary human epidermal cells revealed three sequential modules of cis-elements that specify keratinocyte fate, orchestrated by the TFs JUNB, KLF4, ZNF750, CEBPA, and EHF. Combinatorial deletion of all pairs of these TFs uncovered their epistatic relationships and highlighted genomic co-localization as a basis for synergistic interactions. Thus, Perturb-ATAC is a powerful and general strategy to dissect gene regulatory networks in development and disease.\n\nHighlightsO_LIA new method for simultaneous measurement of CRISPR perturbations and chromatin state in single cells.\nC_LIO_LIPerturb-ATAC reveals regulatory factors that control cis-element accessibility, trans-factor occupancy, and nucleosome positioning.\nC_LIO_LIPerturb-ATAC reveals regulatory modules of coordinated trans-factor activity in B lymphoblasts.\nC_LIO_LIKeratinocyte differentiation is orchestrated by synergistic activities of co-binding TFs on cis-elements.\nC_LI

genomics↗

bioRxiv2017

ciRS-7 exonic sequence is embedded in a long-noncoding RNA locus

ciRS-7 is an intensely studied, highly expressed and conserved circRNA. Essentially nothing is known about its biogenesis, including the location of its promoter. A prevailing assumption has been that ciRS-7 is an exceptional circRNA because it is transcribed from a locus lacking any mature linear RNA transcripts of the same sense. Our interest in the biogenesis of ciRS-7 led us to develop an algorithm to define its promoter. This approach predicted that the human ciRS-7 promoter coincides with that of the long non-coding RNA, LINC00632. We validated this prediction using multiple orthogonal experimental assays. We also used computational approaches and experimental validation to establish that ciRS-7 exonic sequence is embedded in linear transcripts that are flanked by cryptic exons in both human and mouse. Together, this experimental and computational evidence generate a new view of regulation in this locus: (a) ciRS-7 is like other circRNAs, as it is spliced into linear transcripts; (b) expression of ciRS-7 is primarily determined by the chromatin state of LINC00632 promoters; (c) transcription and splicing factors sufficient for ciRS-7 biogenesis are expressed in cells that lack detectable ciRS-7 expression. These findings have significant implications for the study of the regulation and function of ciRS-7, and the analytic framework we developed to jointly analyze RNA-seq and ChlP-seq data reveal the potential for genome-wide discovery of important biological regulation missed in current reference annotations.\n\nAuthor SummarycircRNAs were recently discovered to be a significant product of host gene expression programs but little is known about their transcriptional regulation. Here, we have studied the expression of a well-known circRNA named ciRS-7. ciRS-7 has an unusual function for a circRNA; it is believed to be a miRNA sponge. Previously, ciRS-7 was thought to be transcribed from a locus lacking any mature linear isoforms, unlike all other circular RNAs known to be expressed in human cells. However, we have found this to be false; using a combination of bioinformatic and experimental genetic approaches, in both human and mouse, we discovered that linear transcripts containing the ciRS-7 exonic sequence, linking it to upstream genes. This suggests the potential for additional functional roles of this important locus and provides critical information to begin study on the biogenesis of ciRS-7.

genetics↗