ABSTRACT
Here, we report inducible mosaic animal for perturbation (iMAP), a transgenic platform enabling in situ CRISPR targeting of at least 100 genes in parallel throughout the mouse body. iMAP combines Cre-loxP and CRISPR-Cas9 technologies and utilizes a germline-transmitted transgene carrying a large array of individually floxed, tandemly linked gRNA-coding units. Cre-mediated recombination triggers expression of all the gRNAs in the array but only one of them per cell, converting the mice to mosaic organisms suitable for phenotypic characterization and also for high-throughput derivation of conventional single-gene perturbation lines via breeding. Using gRNA representation as a readout, we mapped a miniature Perturb-Atlas cataloging the perturbations of 90 genes across 39 tissues, which yields rich insights into context-dependent gene functions and provides a glimpse of the potential of iMAP in genome decoding.
Subject(s)
CRISPR-Cas Systems , RNA, Guide, Kinetoplastida , Animals , CRISPR-Cas Systems/genetics , Gene Editing , Genome , Mice , RNA, Guide, Kinetoplastida/genetics , RNA, Guide, Kinetoplastida/metabolism , TransgenesABSTRACT
Live bacterial therapeutics (LBTs) could reverse diseases by engrafting in the gut and providing persistent beneficial functions in the host. However, attempts to functionally manipulate the gut microbiome of conventionally raised (CR) hosts have been unsuccessful because engineered microbial organisms (i.e., chassis) have difficulty in colonizing the hostile luminal environment. In this proof-of-concept study, we use native bacteria as chassis for transgene delivery to impact CR host physiology. Native Escherichia coli bacteria isolated from the stool cultures of CR mice were modified to express functional genes. The reintroduction of these strains induces perpetual engraftment in the intestine. In addition, engineered native E. coli can induce functional changes that affect physiology of and reverse pathology in CR hosts months after administration. Thus, using native bacteria as chassis to "knock in" specific functions allows mechanistic studies of specific microbial activities in the microbiome of CR hosts and enables LBT with curative intent.
Subject(s)
Gastrointestinal Microbiome , Microbiota , Animals , Bacteria/genetics , Escherichia coli/genetics , Gastrointestinal Microbiome/physiology , Mice , TransgenesABSTRACT
The mesencephalic locomotor region (MLR) is a key midbrain center with roles in locomotion. Despite extensive studies and clinical trials aimed at therapy-resistant Parkinson's disease (PD), debate on its function remains. Here, we reveal the existence of functionally diverse neuronal populations with distinct roles in control of body movements. We identify two spatially intermingled glutamatergic populations separable by axonal projections, mouse genetics, neuronal activity profiles, and motor functions. Most spinally projecting MLR neurons encoded the full-body behavior rearing. Loss- and gain-of-function optogenetic perturbation experiments establish a function for these neurons in controlling body extension. In contrast, Rbp4-transgene-positive MLR neurons project in an ascending direction to basal ganglia, preferentially encode the forelimb behaviors handling and grooming, and exhibit a role in modulating movement. Thus, the MLR contains glutamatergic neuronal subpopulations stratified by projection target exhibiting roles in action control not restricted to locomotion.
Subject(s)
Locomotion/physiology , Mesencephalon/anatomy & histology , Animals , Basal Ganglia/metabolism , Behavior, Animal , Female , Integrases/metabolism , Male , Mice, Inbred C57BL , Mice, Transgenic , Neurons/metabolism , Optogenetics , Retinol-Binding Proteins, Plasma/metabolism , Spinal Cord/metabolism , Transgenes , Vesicular Glutamate Transport Protein 2/metabolismABSTRACT
Comprehensively resolving neuronal identities in whole-brain images is a major challenge. We achieve this in C. elegans by engineering a multicolor transgene called NeuroPAL (a neuronal polychromatic atlas of landmarks). NeuroPAL worms share a stereotypical multicolor fluorescence map for the entire hermaphrodite nervous system that resolves all neuronal identities. Neurons labeled with NeuroPAL do not exhibit fluorescence in the green, cyan, or yellow emission channels, allowing the transgene to be used with numerous reporters of gene expression or neuronal dynamics. We showcase three applications that leverage NeuroPAL for nervous-system-wide neuronal identification. First, we determine the brainwide expression patterns of all metabotropic receptors for acetylcholine, GABA, and glutamate, completing a map of this communication network. Second, we uncover changes in cell fate caused by transcription factor mutations. Third, we record brainwide activity in response to attractive and repulsive chemosensory cues, characterizing multimodal coding for these stimuli.
Subject(s)
Atlases as Topic , Brain Mapping , Brain/physiology , Caenorhabditis elegans/physiology , Neurons/physiology , Software , Algorithms , Anatomic Landmarks , Animals , Cell Body/physiology , Cell Lineage , Drosophila/physiology , Mutation/genetics , Nerve Net/physiology , Phenotype , Receptors, Metabotropic Glutamate/metabolism , Receptors, Neurotransmitter/metabolism , Smell/physiology , Taste/physiology , Transcription Factors/metabolism , TransgenesABSTRACT
Replacing or editing disease-causing mutations holds great promise for treating many human diseases. Yet, delivering therapeutic genetic modifiers to specific cells in vivo has been challenging, particularly in large, anatomically distributed tissues such as skeletal muscle. Here, we establish an in vivo strategy to evolve and stringently select capsid variants of adeno-associated viruses (AAVs) that enable potent delivery to desired tissues. Using this method, we identify a class of RGD motif-containing capsids that transduces muscle with superior efficiency and selectivity after intravenous injection in mice and non-human primates. We demonstrate substantially enhanced potency and therapeutic efficacy of these engineered vectors compared to naturally occurring AAV capsids in two mouse models of genetic muscle disease. The top capsid variants from our selection approach show conserved potency for delivery across a variety of inbred mouse strains, and in cynomolgus macaques and human primary myotubes, with transduction dependent on target cell expressed integrin heterodimers.
Subject(s)
Capsid/metabolism , Dependovirus/metabolism , Directed Molecular Evolution , Gene Transfer Techniques , Muscle, Skeletal/metabolism , Amino Acid Sequence , Animals , Capsid/chemistry , Cells, Cultured , Disease Models, Animal , HEK293 Cells , Humans , Integrins/metabolism , Macaca fascicularis , Mice, Inbred BALB C , Mice, Inbred C57BL , Muscle Fibers, Skeletal/metabolism , Muscular Dystrophy, Duchenne/pathology , Muscular Dystrophy, Duchenne/therapy , Myopathies, Structural, Congenital/pathology , Myopathies, Structural, Congenital/therapy , Protein Multimerization , Protein Tyrosine Phosphatases, Non-Receptor/genetics , Protein Tyrosine Phosphatases, Non-Receptor/metabolism , Protein Tyrosine Phosphatases, Non-Receptor/therapeutic use , RNA, Guide, Kinetoplastida/metabolism , Recombination, Genetic/genetics , Species Specificity , TransgenesABSTRACT
In situ transgenesis methods such as viruses and electroporation can rapidly create somatic transgenic mice but lack control over copy number, zygosity, and locus specificity. Here we establish mosaic analysis by dual recombinase-mediated cassette exchange (MADR), which permits stable labeling of mutant cells expressing transgenic elements from precisely defined chromosomal loci. We provide a toolkit of MADR elements for combination labeling, inducible and reversible transgene manipulation, VCre recombinase expression, and transgenesis of human cells. Further, we demonstrate the versatility of MADR by creating glioma models with mixed reporter-identified zygosity or with "personalized" driver mutations from pediatric glioma. MADR is extensible to thousands of existing mouse lines, providing a flexible platform to democratize the generation of somatic mosaic mice. VIDEO ABSTRACT.
Subject(s)
Brain Neoplasms/genetics , Disease Models, Animal , Gene Targeting/methods , Genetic Loci/genetics , Glioma/genetics , Mutagenesis, Insertional/methods , Transgenes/genetics , Animals , Cell Line, Tumor , Female , HEK293 Cells , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neural Stem Cells/metabolism , Recombinases/metabolism , TransfectionABSTRACT
Transcriptional downregulation caused by intronic triplet repeat expansions underlies diseases such as Friedreich's ataxia. This downregulation of gene expression is coupled with epigenetic changes, but the underlying mechanisms are unknown. Here, we show that an intronic GAA/TTC triplet expansion within the IIL1 gene of Arabidopsis thaliana results in accumulation of 24-nt short interfering RNAs (siRNAs) and repressive histone marks at the IIL1 locus, which in turn causes its transcriptional downregulation and an associated phenotype. Knocking down DICER LIKE-3 (DCL3), which produces 24-nt siRNAs, suppressed transcriptional downregulation of IIL1 and the triplet expansion-associated phenotype. Furthermore, knocking down additional components of the RNA-dependent DNA methylation (RdDM) pathway also suppressed both transcriptional downregulation of IIL1 and the repeat expansion-associated phenotype. Thus, our results show that triplet repeat expansions can lead to local siRNA biogenesis, which in turn downregulates transcription through an RdDM-dependent epigenetic modification.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Epigenesis, Genetic , Introns , RNA, Plant/genetics , RNA, Small Interfering/genetics , Ribonuclease III/genetics , Transcription, Genetic , DNA Methylation , DNA Polymerase beta/genetics , Down-Regulation , Gene Expression Regulation, Plant , Genes, Plant , Oligonucleotides, Antisense/genetics , Phenotype , RNA Interference , Transgenes , Trinucleotide Repeat ExpansionABSTRACT
In the auditory system, type I spiral ganglion neurons (SGNs) convey complex acoustic information from inner hair cells (IHCs) to the brainstem. Although SGNs exhibit variation in physiological and anatomical properties, it is unclear which features are endogenous and which reflect input from synaptic partners. Using single-cell RNA sequencing, we derived a molecular classification of mouse type I SGNs comprising three subtypes that express unique combinations of Ca2+ binding proteins, ion channel regulators, guidance molecules, and transcription factors. Based on connectivity and susceptibility to age-related loss, these subtypes correspond to those defined physiologically. Additional intrinsic differences among subtypes and across the tonotopic axis highlight an unexpectedly active role for SGNs in auditory processing. SGN identities emerge postnatally and are disrupted in a mouse model of deafness that lacks IHC-driven activity. These results elucidate the range, nature, and origins of SGN diversity, with implications for treatment of congenital deafness.
Subject(s)
Ear, Inner/physiology , Hair Cells, Auditory, Inner/physiology , Sensory Receptor Cells/physiology , Amino Acid Transport Systems, Acidic/genetics , Animals , Calbindin 2/genetics , Cochlea/physiology , Deafness/genetics , Female , Male , Mice , Mice, Inbred C57BL , Sequence Analysis, RNA , Spiral Ganglion/physiology , Synaptic Transmission , TransgenesABSTRACT
Modern genetic approaches are powerful in providing access to diverse cell types in the brain and facilitating the study of their function. Here, we report a large set of driver and reporter transgenic mouse lines, including 23 new driver lines targeting a variety of cortical and subcortical cell populations and 26 new reporter lines expressing an array of molecular tools. In particular, we describe the TIGRE2.0 transgenic platform and introduce Cre-dependent reporter lines that enable optical physiology, optogenetics, and sparse labeling of genetically defined cell populations. TIGRE2.0 reporters broke the barrier in transgene expression level of single-copy targeted-insertion transgenesis in a wide range of neuronal types, along with additional advantage of a simplified breeding strategy compared to our first-generation TIGRE lines. These novel transgenic lines greatly expand the repertoire of high-precision genetic tools available to effectively identify, monitor, and manipulate distinct cell types in the mouse brain.
Subject(s)
Brain/metabolism , Gene Knockout Techniques/methods , Genes, Reporter , Animals , Brain/cytology , Calcium/metabolism , Cell Line , In Situ Hybridization, Fluorescence , Light , Mice , Mice, Transgenic , Microscopy, Fluorescence , Neurons/metabolism , Optogenetics , RNA, Untranslated/genetics , Transgenes/geneticsABSTRACT
Cells benefit from silencing foreign genetic elements but must simultaneously avoid inactivating endogenous genes. Although chromatin modifications and RNAs contribute to maintenance of silenced states, the establishment of silenced regions will inevitably reflect underlying DNA sequence and/or structure. Here, we demonstrate that a pervasive non-coding DNA feature in Caenorhabditis elegans, characterized by 10-base pair periodic An/Tn-clusters (PATCs), can license transgenes for germline expression within repressive chromatin domains. Transgenes containing natural or synthetic PATCs are resistant to position effect variegation and stochastic silencing in the germline. Among endogenous genes, intron length and PATC-character undergo dramatic changes as orthologs move from active to repressive chromatin over evolutionary time, indicating a dynamic character to the An/Tn periodicity. We propose that PATCs form the basis of a cellular immune system, identifying certain endogenous genes in heterochromatic contexts as privileged while foreign DNA can be suppressed with no requirement for a cellular memory of prior exposure.
Subject(s)
Caenorhabditis elegans/metabolism , DNA, Intergenic/metabolism , Gene Silencing , Animals , Base Composition , Caenorhabditis elegans/genetics , Chromatin , DNA Transposable Elements , DNA, Viral/genetics , Germ Cells/metabolism , Introns , Promoter Regions, Genetic , RNA, Antisense/metabolism , RNA, Messenger/metabolism , TransgenesABSTRACT
T lymphocytes and B lymphocytes integrate activating signals to control the size of their proliferative response. Here we report that such control was achieved by timed changes in the production rate of cell-cycle-regulating proto-oncoprotein Myc, with division cessation occurring when Myc levels fell below a critical threshold. The changing pattern of the level of Myc was not affected by cell division, which identified the regulating mechanism as a cell-intrinsic, heritable temporal controller. Overexpression of Myc in stimulated T cells and B cells did not sustain cell proliferation indefinitely, as a separate 'time-to-die' mechanism, also heritable, was programmed after lymphocyte activation and led to eventual cell loss. Together the two competing cell-intrinsic timed fates created the canonical T cell and B cell immune-response pattern of rapid growth followed by loss of most cells. Furthermore, small changes in these timed processes by regulatory signals, or by oncogenic transformation, acted in synergy to greatly enhance cell numbers over time.
Subject(s)
B-Lymphocytes/physiology , CD8-Positive T-Lymphocytes/physiology , Cell Division , Cell Proliferation/genetics , Immunity, Cellular , Proto-Oncogene Proteins c-myc/metabolism , Animals , Cell Death/genetics , Cell Division/genetics , Cells, Cultured , Gene Expression Regulation , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Mice, Transgenic , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-myc/genetics , Signal Transduction , Transgenes/geneticsABSTRACT
Speciation leads to adaptive changes in organ cellular physiology and creates challenges for studying rare cell-type functions that diverge between humans and mice. Rare cystic fibrosis transmembrane conductance regulator (CFTR)-rich pulmonary ionocytes exist throughout the cartilaginous airways of humans1,2, but limited presence and divergent biology in the proximal trachea of mice has prevented the use of traditional transgenic models to elucidate ionocyte functions in the airway. Here we describe the creation and use of conditional genetic ferret models to dissect pulmonary ionocyte biology and function by enabling ionocyte lineage tracing (FOXI1-CreERT2::ROSA-TG), ionocyte ablation (FOXI1-KO) and ionocyte-specific deletion of CFTR (FOXI1-CreERT2::CFTRL/L). By comparing these models with cystic fibrosis ferrets3,4, we demonstrate that ionocytes control airway surface liquid absorption, secretion, pH and mucus viscosity-leading to reduced airway surface liquid volume and impaired mucociliary clearance in cystic fibrosis, FOXI1-KO and FOXI1-CreERT2::CFTRL/L ferrets. These processes are regulated by CFTR-dependent ionocyte transport of Cl- and HCO3-. Single-cell transcriptomics and in vivo lineage tracing revealed three subtypes of pulmonary ionocytes and a FOXI1-lineage common rare cell progenitor for ionocytes, tuft cells and neuroendocrine cells during airway development. Thus, rare pulmonary ionocytes perform critical CFTR-dependent functions in the proximal airway that are hallmark features of cystic fibrosis airway disease. These studies provide a road map for using conditional genetics in the first non-rodent mammal to address gene function, cell biology and disease processes that have greater evolutionary conservation between humans and ferrets.
Subject(s)
Cystic Fibrosis , Disease Models, Animal , Ferrets , Lung , Transgenes , Animals , Humans , Animals, Genetically Modified , Cell Lineage , Cystic Fibrosis/genetics , Cystic Fibrosis/metabolism , Cystic Fibrosis/pathology , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Ferrets/genetics , Ferrets/physiology , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Lung/cytology , Lung/metabolism , Lung/pathology , Trachea/cytology , Transgenes/geneticsABSTRACT
T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells1-3. Here we developed a clinical-grade approach based on CRISPR-Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRß). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial ( NCT03970382 ). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.
Subject(s)
Cell- and Tissue-Based Therapy , Gene Editing , Neoplasms , Precision Medicine , Receptors, Antigen, T-Cell , T-Lymphocytes , Transgenes , Humans , Antigens, Neoplasm/genetics , Antigens, Neoplasm/immunology , Biopsy , Cell- and Tissue-Based Therapy/adverse effects , Cell- and Tissue-Based Therapy/methods , Cytokine Release Syndrome/complications , Disease Progression , Encephalitis/complications , Gene Knock-In Techniques , Gene Knockout Techniques , Genes, T-Cell Receptor alpha , Genes, T-Cell Receptor beta , Mutation , Neoplasms/complications , Neoplasms/genetics , Neoplasms/immunology , Neoplasms/therapy , Patient Safety , Precision Medicine/adverse effects , Precision Medicine/methods , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Transgenes/genetics , HLA Antigens/immunology , CRISPR-Cas SystemsABSTRACT
Abundant high-molecular-mass hyaluronic acid (HMM-HA) contributes to cancer resistance and possibly to the longevity of the longest-lived rodent-the naked mole-rat1,2. To study whether the benefits of HMM-HA could be transferred to other animal species, we generated a transgenic mouse overexpressing naked mole-rat hyaluronic acid synthase 2 gene (nmrHas2). nmrHas2 mice showed an increase in hyaluronan levels in several tissues, and a lower incidence of spontaneous and induced cancer, extended lifespan and improved healthspan. The transcriptome signature of nmrHas2 mice shifted towards that of longer-lived species. The most notable change observed in nmrHas2 mice was attenuated inflammation across multiple tissues. HMM-HA reduced inflammation through several pathways, including a direct immunoregulatory effect on immune cells, protection from oxidative stress and improved gut barrier function during ageing. These beneficial effects were conferred by HMM-HA and were not specific to the nmrHas2 gene. These findings demonstrate that the longevity mechanism that evolved in the naked mole-rat can be exported to other species, and open new paths for using HMM-HA to improve lifespan and healthspan.
Subject(s)
Healthy Aging , Hyaluronan Synthases , Hyaluronic Acid , Longevity , Mole Rats , Animals , Mice , Hyaluronic Acid/biosynthesis , Hyaluronic Acid/metabolism , Inflammation/genetics , Inflammation/immunology , Inflammation/prevention & control , Mice, Transgenic , Mole Rats/genetics , Longevity/genetics , Longevity/immunology , Longevity/physiology , Hyaluronan Synthases/genetics , Hyaluronan Synthases/metabolism , Healthy Aging/genetics , Healthy Aging/immunology , Healthy Aging/physiology , Transgenes/genetics , Transgenes/physiology , Transcriptome , Neoplasms/genetics , Neoplasms/prevention & control , Oxidative Stress , Geroscience , Rejuvenation/physiologyABSTRACT
Recent human decedent model studies1,2 and compassionate xenograft use3 have explored the promise of porcine organs for human transplantation. To proceed to human studies, a clinically ready porcine donor must be engineered and its xenograft successfully tested in nonhuman primates. Here we describe the design, creation and long-term life-supporting function of kidney grafts from a genetically engineered porcine donor transplanted into a cynomolgus monkey model. The porcine donor was engineered to carry 69 genomic edits, eliminating glycan antigens, overexpressing human transgenes and inactivating porcine endogenous retroviruses. In vitro functional analyses showed that the edited kidney endothelial cells modulated inflammation to an extent that was indistinguishable from that of human endothelial cells, suggesting that these edited cells acquired a high level of human immune compatibility. When transplanted into cynomolgus monkeys, the kidneys with three glycan antigen knockouts alone experienced poor graft survival, whereas those with glycan antigen knockouts and human transgene expression demonstrated significantly longer survival time, suggesting the benefit of human transgene expression in vivo. These results show that preclinical studies of renal xenotransplantation could be successfully conducted in nonhuman primates and bring us closer to clinical trials of genetically engineered porcine renal grafts.
Subject(s)
Graft Rejection , Kidney Transplantation , Macaca fascicularis , Swine , Transplantation, Heterologous , Animals , Humans , Animals, Genetically Modified , Endothelial Cells/immunology , Endothelial Cells/metabolism , Graft Rejection/immunology , Graft Rejection/prevention & control , Kidney Transplantation/methods , Polysaccharides/deficiency , Swine/genetics , Transplantation, Heterologous/methods , Transgenes/geneticsABSTRACT
Spatially charting molecular cell types at single-cell resolution across the 3D volume is critical for illustrating the molecular basis of brain anatomy and functions. Single-cell RNA sequencing has profiled molecular cell types in the mouse brain1,2, but cannot capture their spatial organization. Here we used an in situ sequencing method, STARmap PLUS3,4, to profile 1,022 genes in 3D at a voxel size of 194 × 194 × 345 nm3, mapping 1.09 million high-quality cells across the adult mouse brain and spinal cord. We developed computational pipelines to segment, cluster and annotate 230 molecular cell types by single-cell gene expression and 106 molecular tissue regions by spatial niche gene expression. Joint analysis of molecular cell types and molecular tissue regions enabled a systematic molecular spatial cell-type nomenclature and identification of tissue architectures that were undefined in established brain anatomy. To create a transcriptome-wide spatial atlas, we integrated STARmap PLUS measurements with a published single-cell RNA-sequencing atlas1, imputing single-cell expression profiles of 11,844 genes. Finally, we delineated viral tropisms of a brain-wide transgene delivery tool, AAV-PHP.eB5,6. Together, this annotated dataset provides a single-cell resource that integrates the molecular spatial atlas, brain anatomy and the accessibility to genetic manipulation of the mammalian central nervous system.
Subject(s)
Central Nervous System , Imaging, Three-Dimensional , Single-Cell Analysis , Transcriptome , Animals , Mice , Brain/anatomy & histology , Brain/cytology , Brain/metabolism , Central Nervous System/anatomy & histology , Central Nervous System/cytology , Central Nervous System/metabolism , Single-Cell Analysis/methods , Spinal Cord/anatomy & histology , Spinal Cord/cytology , Spinal Cord/metabolism , Transcriptome/genetics , Single-Cell Gene Expression Analysis , Viral Tropism , Datasets as Topic , Transgenes/genetics , Imaging, Three-Dimensional/methodsABSTRACT
Viral RNA-dependent RNA polymerases (RdRps) are a target for broad-spectrum antiviral therapeutic agents. Recently, we demonstrated that incorporation of the T-1106 triphosphate, a pyrazine-carboxamide ribonucleotide, into nascent RNA increases pausing and backtracking by the poliovirus RdRp. Here, by monitoring enterovirus A-71 RdRp dynamics during RNA synthesis using magnetic tweezers, we identify the "backtracked" state as an intermediate used by the RdRp for copy-back RNA synthesis and homologous recombination. Cell-based assays and RNA sequencing (RNA-seq) experiments further demonstrate that the pyrazine-carboxamide ribonucleotide stimulates these processes during infection. These results suggest that pyrazine-carboxamide ribonucleotides do not induce lethal mutagenesis or chain termination but function by promoting template switching and formation of defective viral genomes. We conclude that RdRp-catalyzed intra- and intermolecular template switching can be induced by pyrazine-carboxamide ribonucleotides, defining an additional mechanistic class of antiviral ribonucleotides with potential for broad-spectrum activity.
Subject(s)
Pyrazines/chemistry , RNA Viruses/genetics , RNA, Viral/genetics , RNA-Dependent RNA Polymerase/genetics , Recombination, Genetic , Ribonucleotides/chemistry , Animals , Antiviral Agents , Catalysis , Cells, Cultured , Genetic Techniques , Genome , Genome, Viral , Homologous Recombination , Humans , Kinetics , Mice , Mice, Transgenic , Molecular Dynamics Simulation , Mutagenesis , Nucleotides/genetics , Protein Conformation , RNA/chemistry , RNA-Dependent RNA Polymerase/metabolism , RNA-Seq , Transgenes , VirulenceABSTRACT
Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, preclinical and clinical research with PARPi has revealed multiple resistance mechanisms, highlighting the need for identification of novel functional biomarkers and combination treatment strategies. Functional genetic screens performed in cells and organoids that acquired resistance to PARPi by loss of 53BP1 identified loss of LIG3 as an enhancer of PARPi toxicity in BRCA1-deficient cells. Enhancement of PARPi toxicity by LIG3 depletion is dependent on BRCA1 deficiency but independent of the loss of 53BP1 pathway. Mechanistically, we show that LIG3 loss promotes formation of MRE11-mediated post-replicative ssDNA gaps in BRCA1-deficient and BRCA1/53BP1 double-deficient cells exposed to PARPi, leading to an accumulation of chromosomal abnormalities. LIG3 depletion also enhances efficacy of PARPi against BRCA1-deficient mammary tumors in mice, suggesting LIG3 as a potential therapeutic target.
Subject(s)
BRCA1 Protein/genetics , DNA Ligase ATP/genetics , DNA, Single-Stranded , MRE11 Homologue Protein/genetics , Ovarian Neoplasms/metabolism , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly-ADP-Ribose Binding Proteins/genetics , Triple Negative Breast Neoplasms/metabolism , Tumor Suppressor p53-Binding Protein 1/genetics , Animals , Biopsy , CRISPR-Cas Systems , Cell Line , Cell Nucleus/metabolism , Cell Proliferation , Chromosome Aberrations , DNA Damage , DNA Ligase ATP/metabolism , Female , Humans , Lentivirus/genetics , Mammary Neoplasms, Animal , Mice , Mutation , Poly-ADP-Ribose Binding Proteins/metabolism , RNA, Small Interfering/metabolism , TransgenesABSTRACT
All life forms defend their genome against DNA invasion. Eukaryotic cells recognize incoming DNA and limit its transcription through repressive chromatin modifications. The human silencing hub (HUSH) complex transcriptionally represses long interspersed element-1 retrotransposons (L1s) and retroviruses through histone H3 lysine 9 trimethylation (H3K9me3)1-3. How HUSH recognizes and initiates silencing of these invading genetic elements is unknown. Here we show that HUSH is able to recognize and transcriptionally repress a broad range of long, intronless transgenes. Intron insertion into HUSH-repressed transgenes counteracts repression, even in the absence of intron splicing. HUSH binds transcripts from the target locus, prior to and independent of H3K9me3 deposition, and target transcription is essential for both initiation and propagation of HUSH-mediated H3K9me3. Genomic data reveal how HUSH binds and represses a subset of endogenous intronless genes generated through retrotransposition of cellular mRNAs. Thus intronless cDNA-the hallmark of reverse transcription-provides a versatile way to distinguish invading retroelements from host genes and enables HUSH to protect the genome from 'non-self' DNA, despite there being no previous exposure to the invading element. Our findings reveal the existence of a transcription-dependent genome-surveillance system and explain how it provides immediate protection against newly acquired elements while avoiding inappropriate repression of host genes.
Subject(s)
Gene Silencing , Long Interspersed Nucleotide Elements , Retroelements , Histones/genetics , Humans , Introns , Long Interspersed Nucleotide Elements/genetics , Methylation , Retroelements/genetics , TransgenesABSTRACT
Entomopathogenic nematodes are widely used as biopesticides1,2. Their insecticidal activity depends on symbiotic bacteria such as Photorhabdus luminescens, which produces toxin complex (Tc) toxins as major virulence factors3-6. No protein receptors are known for any Tc toxins, which limits our understanding of their specificity and pathogenesis. Here we use genome-wide CRISPR-Cas9-mediated knockout screening in Drosophila melanogaster S2R+ cells and identify Visgun (Vsg) as a receptor for an archetypal P. luminescens Tc toxin (pTc). The toxin recognizes the extracellular O-glycosylated mucin-like domain of Vsg that contains high-density repeats of proline, threonine and serine (HD-PTS). Vsg orthologues in mosquitoes and beetles contain HD-PTS and can function as pTc receptors, whereas orthologues without HD-PTS, such as moth and human versions, are not pTc receptors. Vsg is expressed in immune cells, including haemocytes and fat body cells. Haemocytes from Vsg knockout Drosophila are resistant to pTc and maintain phagocytosis in the presence of pTc, and their sensitivity to pTc is restored through the transgenic expression of mosquito Vsg. Last, Vsg knockout Drosophila show reduced bacterial loads and lethality from P. luminescens infection. Our findings identify a proteinaceous Tc toxin receptor, reveal how Tc toxins contribute to P. luminescens pathogenesis, and establish a genome-wide CRISPR screening approach for investigating insecticidal toxins and pathogens.