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Molecular Cell Oct 2021Compact and versatile CRISPR-Cas systems will enable genome engineering applications through high-efficiency delivery in a wide variety of contexts. Here, we create an... (Comparative Study)
Comparative Study
Compact and versatile CRISPR-Cas systems will enable genome engineering applications through high-efficiency delivery in a wide variety of contexts. Here, we create an efficient miniature Cas system (CasMINI) engineered from the type V-F Cas12f (Cas14) system by guide RNA and protein engineering, which is less than half the size of currently used CRISPR systems (Cas9 or Cas12a). We demonstrate that CasMINI can drive high levels of gene activation (up to thousands-fold increases), while the natural Cas12f system fails to function in mammalian cells. We show that the CasMINI system has comparable activities to Cas12a for gene activation, is highly specific, and allows robust base editing and gene editing. We expect that CasMINI can be broadly useful for cell engineering and gene therapy applications ex vivo and in vivo.
Topics: CRISPR-Associated Proteins; CRISPR-Cas Systems; Clustered Regularly Interspaced Short Palindromic Repeats; Gene Editing; Genes, Reporter; Green Fluorescent Proteins; HEK293 Cells; Humans; Mutation; Promoter Regions, Genetic; Protein Engineering; RNA, Guide, CRISPR-Cas Systems; Transcriptional Activation
PubMed: 34480847
DOI: 10.1016/j.molcel.2021.08.008 -
Cell Host & Microbe May 2020The ongoing pandemic of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), underscores the urgency to develop experimental systems for...
The ongoing pandemic of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), underscores the urgency to develop experimental systems for studying this virus and identifying countermeasures. We report a reverse genetic system for SARS-CoV-2. Seven complimentary DNA (cDNA) fragments spanning the SARS-CoV-2 genome were assembled into a full-genome cDNA. RNA transcribed from the full-genome cDNA was highly infectious after electroporation into cells, producing 2.9 × 10 plaque-forming unit (PFU)/mL of virus. Compared with a clinical isolate, the infectious-clone-derived SARS-CoV-2 (icSARS-CoV-2) exhibited similar plaque morphology, viral RNA profile, and replication kinetics. Additionally, icSARS-CoV-2 retained engineered molecular markers and did not acquire other mutations. We generated a stable mNeonGreen SARS-CoV-2 (icSARS-CoV-2-mNG) by introducing this reporter gene into ORF7 of the viral genome. icSARS-CoV-2-mNG was successfully used to evaluate the antiviral activities of interferon (IFN). Collectively, the reverse genetic system and reporter virus provide key reagents to study SARS-CoV-2 and develop countermeasures.
Topics: Animals; Antiviral Agents; Betacoronavirus; COVID-19; Chlorocebus aethiops; Clone Cells; Coronavirus Infections; DNA, Complementary; Genes, Reporter; Genome, Viral; Interferons; Organisms, Genetically Modified; Pandemics; Pneumonia, Viral; RNA, Viral; SARS-CoV-2; Vero Cells; Virus Replication
PubMed: 32289263
DOI: 10.1016/j.chom.2020.04.004 -
Nature Communications Jan 2021Endothelial cells play a critical role in the adaptation of tissues to injury. Tissue ischemia induced by infarction leads to profound changes in endothelial cell...
Endothelial cells play a critical role in the adaptation of tissues to injury. Tissue ischemia induced by infarction leads to profound changes in endothelial cell functions and can induce transition to a mesenchymal state. Here we explore the kinetics and individual cellular responses of endothelial cells after myocardial infarction by using single cell RNA sequencing. This study demonstrates a time dependent switch in endothelial cell proliferation and inflammation associated with transient changes in metabolic gene signatures. Trajectory analysis reveals that the majority of endothelial cells 3 to 7 days after myocardial infarction acquire a transient state, characterized by mesenchymal gene expression, which returns to baseline 14 days after injury. Lineage tracing, using the Cdh5-CreERT2;mT/mG mice followed by single cell RNA sequencing, confirms the transient mesenchymal transition and reveals additional hypoxic and inflammatory signatures of endothelial cells during early and late states after injury. These data suggest that endothelial cells undergo a transient mes-enchymal activation concomitant with a metabolic adaptation within the first days after myocardial infarction but do not acquire a long-term mesenchymal fate. This mesenchymal activation may facilitate endothelial cell migration and clonal expansion to regenerate the vascular network.
Topics: Animals; Cell Movement; Cell Plasticity; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Endothelial Cells; Endothelium; Epithelial-Mesenchymal Transition; Genes, Reporter; Human Umbilical Vein Endothelial Cells; Humans; Luminescent Proteins; Male; Mice; Mice, Transgenic; Myocardial Infarction; Myocardium; RNA-Seq; Single-Cell Analysis
PubMed: 33514719
DOI: 10.1038/s41467-021-20905-1 -
Nature Genetics Feb 2023Massively parallel reporter gene assays are key tools in regulatory genomics but cannot be used to identify cell-type-specific regulatory elements without performing...
Massively parallel reporter gene assays are key tools in regulatory genomics but cannot be used to identify cell-type-specific regulatory elements without performing assays serially across different cell types. To address this problem, we developed a single-cell massively parallel reporter assay (scMPRA) to measure the activity of libraries of cis-regulatory sequences (CRSs) across multiple cell types simultaneously. We assayed a library of core promoters in a mixture of HEK293 and K562 cells and showed that scMPRA is a reproducible, highly parallel, single-cell reporter gene assay that detects cell-type-specific cis-regulatory activity. We then measured a library of promoter variants across multiple cell types in live mouse retinas and showed that subtle genetic variants can produce cell-type-specific effects on cis-regulatory activity. We anticipate that scMPRA will be widely applicable for studying the role of CRSs across diverse cell types.
Topics: Animals; Humans; Mice; Gene Library; Genes, Reporter; HEK293 Cells; Promoter Regions, Genetic; Retina
PubMed: 36635387
DOI: 10.1038/s41588-022-01278-7 -
Nature Apr 2023Human gene expression is regulated by more than 2,000 transcription factors and chromatin regulators. Effector domains within these proteins can activate or repress...
Human gene expression is regulated by more than 2,000 transcription factors and chromatin regulators. Effector domains within these proteins can activate or repress transcription. However, for many of these regulators we do not know what type of effector domains they contain, their location in the protein, their activation and repression strengths, and the sequences that are necessary for their functions. Here, we systematically measure the effector activity of more than 100,000 protein fragments tiling across most chromatin regulators and transcription factors in human cells (2,047 proteins). By testing the effect they have when recruited at reporter genes, we annotate 374 activation domains and 715 repression domains, roughly 80% of which are new and have not been previously annotated. Rational mutagenesis and deletion scans across all the effector domains reveal aromatic and/or leucine residues interspersed with acidic, proline, serine and/or glutamine residues are necessary for activation domain activity. Furthermore, most repression domain sequences contain sites for small ubiquitin-like modifier (SUMO)ylation, short interaction motifs for recruiting corepressors or are structured binding domains for recruiting other repressive proteins. We discover bifunctional domains that can both activate and repress, some of which dynamically split a cell population into high- and low-expression subpopulations. Our systematic annotation and characterization of effector domains provide a rich resource for understanding the function of human transcription factors and chromatin regulators, engineering compact tools for controlling gene expression and refining predictive models of effector domain function.
Topics: Humans; Chromatin; Genes, Reporter; Mutagenesis; Transcription Factors; Transcription, Genetic; Protein Domains; Repressor Proteins; Sumoylation; Gene Expression Regulation
PubMed: 37020022
DOI: 10.1038/s41586-023-05906-y -
Nature Communications Nov 2022The progressive decline of physiological function and the increased risk of age-related diseases challenge healthy aging. Multiple anti-aging manipulations, such as...
The progressive decline of physiological function and the increased risk of age-related diseases challenge healthy aging. Multiple anti-aging manipulations, such as senolytics, have proven beneficial for health; however, the biomarkers that label in vivo senescence at systemic levels are lacking, thus hindering anti-aging applications. In this study, we generate a Glb1‒Glb1-2A-mCherry (GAC) reporter allele at the Glb1 gene locus, which encodes lysosomal β-galactosidase-an enzyme elevated in tissues of old mice. A linear correlation between GAC signal and chronological age is established in a cohort of middle-aged (9 to 13 months) Glb1 mice. The high GAC signal is closely associated with cardiac hypertrophy and a shortened lifespan. Moreover, the GAC signal is exponentially increased in pathological senescence induced by bleomycin in the lung. Senolytic dasatinib and quercetin (D + Q) reduce GAC signal in bleomycin treated mice. Thus, the Glb1-2A-mCherry reporter mice monitors systemic aging and function decline, predicts lifespan, and may facilitate the understanding of aging mechanisms and help in the development of anti-aging interventions.
Topics: Animals; Mice; Aging; Bleomycin; Cellular Senescence; Dasatinib; Longevity; Genes, Reporter; Glycoside Hydrolases
PubMed: 36396643
DOI: 10.1038/s41467-022-34801-9 -
International Journal of Molecular... Aug 2023The centrosome of mammalian cells is in constant movement and its motion plays a part in cell differentiation and cell division. The purpose of this study was to...
The centrosome of mammalian cells is in constant movement and its motion plays a part in cell differentiation and cell division. The purpose of this study was to establish the involvement of the TUBG meshwork in centrosomal motility. In live cells, we used a monomeric red-fluorescence-protein-tagged gene and a green-fluorescence-protein-tagged gene for labeling the centrosome and the TUBG1 meshwork, respectively. We found that centrosome movements occurred in cellular sites rich in GTPase TUBG1 and single-guide RNA mediated a reduction in the expression of TUBG1, altering the motility pattern of centrosomes. We propose that the TUBG1 meshwork enables the centrosomes to move by providing them with an interacting platform that mediates positional changes. These findings uncover a novel regulatory mechanism that controls the behavior of centrosomes.
Topics: Animals; Cell Differentiation; Centrosome; Genes, Reporter; Mammals; Humans; Tubulin
PubMed: 37685969
DOI: 10.3390/ijms241713154 -
International Journal of Molecular... Jul 2022The use of molecular imaging technologies for brain imaging can not only play an important supporting role in disease diagnosis and treatment but can also be used to... (Review)
Review
The use of molecular imaging technologies for brain imaging can not only play an important supporting role in disease diagnosis and treatment but can also be used to deeply study brain functions. Recently, with the support of reporter gene technology, optical imaging has achieved a breakthrough in brain function studies at the molecular level. Reporter gene technology based on traditional clinical imaging modalities is also expanding. By benefiting from the deeper imaging depths and wider imaging ranges now possible, these methods have led to breakthroughs in preclinical and clinical research. This article focuses on the applications of magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), and positron emission tomography (PET) reporter gene technologies for use in brain imaging. The tracking of cell therapies and gene therapies is the most successful and widely used application of these techniques. Meanwhile, breakthroughs have been achieved in the research and development of reporter genes and their imaging probe pairs with respect to brain function research. This paper introduces the imaging principles and classifications of the reporter gene technologies of these imaging modalities, lists the relevant brain imaging applications, reviews their characteristics, and discusses the opportunities and challenges faced by clinical imaging modalities based on reporter gene technology. The conclusion is provided in the last section.
Topics: Brain; Genes, Reporter; Magnetic Resonance Imaging; Neuroimaging; Positron-Emission Tomography; Tomography, Emission-Computed, Single-Photon; Tomography, X-Ray Computed
PubMed: 35955578
DOI: 10.3390/ijms23158443 -
Journal of Nuclear Medicine : Official... Dec 2019The first reporter systems were developed in the early 1980s and were based on measuring the activity of an enzyme-as a surrogate measure of promoter-driven... (Review)
Review
The first reporter systems were developed in the early 1980s and were based on measuring the activity of an enzyme-as a surrogate measure of promoter-driven transcriptional activity-which is now known as a reporter gene system. The initial objective and application of reporter techniques was to analyze the activity of a specific promoter (namely, the expression of a gene that is under the regulation of the specific promoter that is linked to the reporter gene). This system allows visualization of specific promoter activity with great sensitivity. In general, there are 2 classes of reporter systems: constitutively expressed (always-on) reporter constructs used for cell tracking, and inducible reporter systems sensitive to endogenous signaling molecules and transcription factors that characterize specific tissues, tumors, or signaling pathways.This review traces the development of different reporter systems, using and proteins as well as reporter systems. The development and application of reporter systems is the focus of this review. The question at the end of the review is whether the "promise" of reporter gene imaging has been realized. What is required for moving forward with reporter systems, and what is required for successful translation to clinical applications?
Topics: Animals; Genes, Reporter; Humans; Molecular Imaging; Radionuclide Imaging
PubMed: 31792128
DOI: 10.2967/jnumed.118.220004 -
Viruses Sep 2020Flaviviruses are significant human pathogens that cause frequent emerging and reemerging epidemics around the world. Better molecular tools for studying, diagnosing, and... (Review)
Review
Flaviviruses are significant human pathogens that cause frequent emerging and reemerging epidemics around the world. Better molecular tools for studying, diagnosing, and treating these diseases are needed. Reporter viruses represent potent tools to fill this gap but have been hindered by genetic instability. Recent advances have overcome these hurdles, opening the way for increased use of stable reporter flaviviruses to diagnose infections, screen and study antiviral compounds, and serve as potential vaccine vectors.
Topics: Animals; Flavivirus; Genes, Reporter; Genetic Engineering; Genome, Viral; Humans; RNA, Viral; Virus Replication
PubMed: 32992987
DOI: 10.3390/v12101082