RESUMO
DNA replication and transcription occur in all living cells across all domains of life. Both essential processes occur simultaneously on the same template, leading to conflicts between the macromolecular machines that perform these functions. Numerous studies over the past few decades demonstrate that this is an inevitable problem in both prokaryotic and eukaryotic cells. We have learned that conflicts lead to replication fork reversal, breaks in the DNA, R-loop formation, topological stress, and mutagenesis and can ultimately impact evolution. Recent studies have also provided insight into the various mechanisms that mitigate, resolve, and allow tolerance of conflicts and how conflicts result in pathological consequences across divergent species. In this review, we summarize our current knowledge regarding the outcomes of the encounters between replication and transcription machineries and explore how these clashes are dealt with across species.
Assuntos
Replicação do DNA , Transcrição Gênica , Humanos , Animais , Cromossomos/metabolismo , Cromossomos/genética , Cromossomos/química , Estruturas R-Loop , DNA/metabolismo , DNA/genética , DNA/químicaRESUMO
Transcription-coupled repair (TCR), discovered as preferential nucleotide excision repair of UV-induced cyclobutane pyrimidine dimers located in transcribed mammalian genes compared to those in nontranscribed regions of the genome, is defined as faster repair of the transcribed strand versus the nontranscribed strand in transcribed genes. The phenomenon, universal in model organisms including Escherichia coli, yeast, Arabidopsis, mice, and humans, involves a translocase that interacts with both RNA polymerase stalled at damage in the transcribed strand and nucleotide excision repair proteins to accelerate repair. Drosophila, a notable exception, exhibits TCR but lacks an obvious TCR translocase. Mutations inactivating TCR genes cause increased damage-induced mutagenesis in E. coli and severe neurological and UV sensitivity syndromes in humans. To date, only E. coli TCR has been reconstituted in vitro with purified proteins. Detailed investigations of TCR using genome-wide next-generation sequencing methods, cryo-electron microscopy, single-molecule analysis, and other approaches have revealed fascinating mechanisms.
Assuntos
Escherichia coli , Transcrição Gênica , Humanos , Animais , Camundongos , Escherichia coli/genética , Escherichia coli/metabolismo , Microscopia Crioeletrônica , Reparo do DNA , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Mamíferos/genéticaRESUMO
CD4+ T cells play fundamental roles in orchestrating immune responses and tissue homeostasis. However, our inability to associate peptide human leukocyte antigen class-II (HLA-II) complexes with their cognate T cell receptors (TCRs) in an unbiased manner has hampered our understanding of CD4+ T cell function and role in pathologies. Here, we introduce TScan-II, a highly sensitive genome-scale CD4+ antigen discovery platform. This platform seamlessly integrates the endogenous HLA-II antigen-processing machinery in synthetic antigen-presenting cells and TCR signaling in T cells, enabling the simultaneous screening of multiple HLAs and TCRs. Leveraging genome-scale human, virome, and epitope mutagenesis libraries, TScan-II facilitates de novo antigen discovery and deep exploration of TCR specificity. We demonstrate TScan-II's potential for basic and translational research by identifying a non-canonical antigen for a cancer-reactive CD4+ T cell clone. Additionally, we identified two antigens for clonally expanded CD4+ T cells in Sjögren's disease, which bind distinct HLAs and are expressed in HLA-II-positive ductal cells within affected salivary glands.
Assuntos
Linfócitos T CD4-Positivos , Epitopos de Linfócito T , Humanos , Células Apresentadoras de Antígenos , Antígenos CD4/metabolismo , Antígenos HLA/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linhagem Celular , Genoma HumanoRESUMO
Our current view of how DNA-based genomes are efficiently and accurately replicated continues to evolve as new details emerge on the presence of ribonucleotides in DNA. Ribonucleotides are incorporated during eukaryotic DNA replication at rates that make them the most common noncanonical nucleotide placed into the nuclear genome, they are efficiently repaired, and their removal impacts genome integrity. This review focuses on three aspects of this subject: the incorporation of ribonucleotides into the eukaryotic nuclear genome during replication by B-family DNA replicases, how these ribonucleotides are removed, and the consequences of their presence or removal for genome stability and disease.
Assuntos
Replicação do DNA , Instabilidade Genômica , Ribonucleotídeos , DNA/genética , DNA/metabolismo , Reparo do DNA , Eucariotos/genética , Eucariotos/metabolismo , Nucleotidiltransferases/genética , Ribonucleotídeos/genética , Ribonucleotídeos/metabolismoRESUMO
Bacterial cytoplasmic membrane vesicles provide a unique experimental system for studying active transport. Vesicles are prepared by lysis of osmotically sensitized cells (i.e., protoplasts or spheroplasts) and comprise osmotically intact, unit-membrane-bound sacs that are approximately 0.5-1.0 µm in diameter and devoid of internal structure. Their metabolic activities are restricted to those provided by the enzymes of the membrane itself, and each vesicle is functional. The energy source for accumulation of a particular substrate can be determined by studying which compounds or experimental conditions drive solute accumulation, and metabolic conversion of the transported substrate or the energy source is minimal. These properties of the vesicle system constitute a considerable advantage over intact cells, as the system provides clear definition of the reactions involved in the transport process. This discussion is not intended as a general review but is concerned with respiration-dependent active transport in membrane vesicles from Escherichia coli. Emphasis is placed on experimental observations demonstrating that respiratory energy is converted primarily into work in the form of a solute concentration gradient that is driven by a proton electrochemical gradient, as postulated by the chemiosmotic theory of Peter Mitchell.
Assuntos
Vesículas Citoplasmáticas/metabolismo , Escherichia coli/metabolismo , Biologia Molecular/história , Transporte Biológico , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Membrana Celular/efeitos dos fármacos , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , História do Século XX , História do Século XXI , Humanos , Ácido Láctico/metabolismo , Masculino , Estados UnidosRESUMO
Genomic DNA is susceptible to endogenous and environmental stresses that modify DNA structure and its coding potential. Correspondingly, cells have evolved intricate DNA repair systems to deter changes to their genetic material. Base excision DNA repair involves a number of enzymes and protein cofactors that hasten repair of damaged DNA bases. Recent advances have identified macromolecular complexes that assemble at the DNA lesion and mediate repair. The repair of base lesions generally requires five enzymatic activities: glycosylase, endonuclease, lyase, polymerase, and ligase. The protein cofactors and mechanisms for coordinating the sequential enzymatic steps of repair are being revealed through a range of experimental approaches. We discuss the enzymes and protein cofactors involved in eukaryotic base excision repair, emphasizing the challenge of integrating findings from multiple methodologies. The results provide an opportunity to assimilate biochemical findings with cell-based assays to uncover new insights into this deceptively complex repair pathway.
Assuntos
DNA Glicosilases/química , DNA Polimerase Dirigida por DNA/química , DNA/química , Endonucleases/química , Genoma , Ligases/química , Liases/química , DNA/metabolismo , DNA/ultraestrutura , Dano ao DNA , DNA Glicosilases/metabolismo , DNA Glicosilases/ultraestrutura , Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/ultraestrutura , Endonucleases/metabolismo , Endonucleases/ultraestrutura , Eucariotos/genética , Eucariotos/metabolismo , Células Eucarióticas/citologia , Células Eucarióticas/enzimologia , Instabilidade Genômica , Humanos , Ligases/metabolismo , Ligases/ultraestrutura , Liases/metabolismo , Liases/ultraestrutura , Modelos Moleculares , Mutagênese , Conformação de Ácido Nucleico , Conformação ProteicaRESUMO
Whole-genome-sequencing (WGS) of human tumors has revealed distinct mutation patterns that hint at the causative origins of cancer. We examined mutational signatures in 324 WGS human-induced pluripotent stem cells exposed to 79 known or suspected environmental carcinogens. Forty-one yielded characteristic substitution mutational signatures. Some were similar to signatures found in human tumors. Additionally, six agents produced double-substitution signatures and eight produced indel signatures. Investigating mutation asymmetries across genome topography revealed fully functional mismatch and transcription-coupled repair pathways. DNA damage induced by environmental mutagens can be resolved by disparate repair and/or replicative pathways, resulting in an assortment of signature outcomes even for a single agent. This compendium of experimentally induced mutational signatures permits further exploration of roles of environmental agents in cancer etiology and underscores how human stem cell DNA is directly vulnerable to environmental agents. VIDEO ABSTRACT.
Assuntos
Carcinógenos Ambientais/classificação , Neoplasias/genética , Carcinógenos Ambientais/efeitos adversos , Dano ao DNA/genética , Análise Mutacional de DNA/métodos , Reparo do DNA/genética , Replicação do DNA , Perfil Genético , Genoma Humano/genética , Humanos , Mutação INDEL/genética , Mutagênese , Mutação/genética , Células-Tronco Pluripotentes/metabolismo , Sequenciamento Completo do Genoma/métodosRESUMO
Multiple signatures of somatic mutations have been identified in cancer genomes. Exome sequences of 1,001 human cancer cell lines and 577 xenografts revealed most common mutational signatures, indicating past activity of the underlying processes, usually in appropriate cancer types. To investigate ongoing patterns of mutational-signature generation, cell lines were cultured for extended periods and subsequently DNA sequenced. Signatures of discontinued exposures, including tobacco smoke and ultraviolet light, were not generated in vitro. Signatures of normal and defective DNA repair and replication continued to be generated at roughly stable mutation rates. Signatures of APOBEC cytidine deaminase DNA-editing exhibited substantial fluctuations in mutation rate over time with episodic bursts of mutations. The initiating factors for the bursts are unclear, although retrotransposon mobilization may contribute. The examined cell lines constitute a resource of live experimental models of mutational processes, which potentially retain patterns of activity and regulation operative in primary human cancers.
Assuntos
Desaminases APOBEC/genética , Neoplasias/genética , Desaminases APOBEC/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , DNA/metabolismo , Análise Mutacional de DNA/métodos , Bases de Dados Genéticas , Exoma , Genoma Humano/genética , Xenoenxertos , Humanos , Mutagênese , Mutação/genética , Taxa de Mutação , Retroelementos , Sequenciamento do Exoma/métodosRESUMO
T cell recognition of specific antigens mediates protection from pathogens and controls neoplasias, but can also cause autoimmunity. Our knowledge of T cell antigens and their implications for human health is limited by the technical limitations of T cell profiling technologies. Here, we present T-Scan, a high-throughput platform for identification of antigens productively recognized by T cells. T-Scan uses lentiviral delivery of antigen libraries into cells for endogenous processing and presentation on major histocompatibility complex (MHC) molecules. Target cells functionally recognized by T cells are isolated using a reporter for granzyme B activity, and the antigens mediating recognition are identified by next-generation sequencing. We show T-Scan correctly identifies cognate antigens of T cell receptors (TCRs) from viral and human genome-wide libraries. We apply T-Scan to discover new viral antigens, perform high-resolution mapping of TCR specificity, and characterize the reactivity of a tumor-derived TCR. T-Scan is a powerful approach for studying T cell responses.
Assuntos
Antígenos de Neoplasias/imunologia , Epitopos de Linfócito T/imunologia , Genes MHC Classe I/imunologia , Antígenos HLA/imunologia , Proteínas de Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Apresentação de Antígeno/imunologia , Antígenos de Neoplasias/genética , Doadores de Sangue , Linfócitos T CD8-Positivos/metabolismo , Feminino , Técnicas de Inativação de Genes , Genes MHC Classe I/genética , Granzimas/metabolismo , Células HEK293 , Antígenos HLA/genética , Humanos , Proteínas de Neoplasias/genética , Transdução Genética , TransfecçãoRESUMO
Flavin-dependent halogenases (FDHs) catalyze the halogenation of organic substrates by coordinating reactions of reduced flavin, molecular oxygen, and chloride. Targeted and random mutagenesis of these enzymes have been used to both understand and alter their reactivity. These studies have led to insights into residues essential for catalysis and FDH variants with improved stability, expanded substrate scope, and altered site selectivity. Mutations throughout FDH structures have contributed to all of these advances. More recent studies have sought to rationalize the impact of these mutations on FDH function and to identify new FDHs to deepen our understanding of this enzyme class and to expand their utility for biocatalytic applications.
Assuntos
Flavinas/metabolismo , Halogenação/genética , Halogenação/fisiologia , Oxirredutases/genética , Oxirredutases/metabolismo , Biocatálise , Domínio Catalítico/genética , Evolução Molecular Direcionada , Desenho de Fármacos , Estabilidade Enzimática/genética , Hidrocarbonetos Halogenados/química , Hidrocarbonetos Halogenados/metabolismo , Redes e Vias Metabólicas , Modelos Moleculares , Mutagênese , Oxirredutases/química , Especificidade por SubstratoRESUMO
The urea cycle (UC) is the main pathway by which mammals dispose of waste nitrogen. We find that specific alterations in the expression of most UC enzymes occur in many tumors, leading to a general metabolic hallmark termed "UC dysregulation" (UCD). UCD elicits nitrogen diversion toward carbamoyl-phosphate synthetase2, aspartate transcarbamylase, and dihydrooratase (CAD) activation and enhances pyrimidine synthesis, resulting in detectable changes in nitrogen metabolites in both patient tumors and their bio-fluids. The accompanying excess of pyrimidine versus purine nucleotides results in a genomic signature consisting of transversion mutations at the DNA, RNA, and protein levels. This mutational bias is associated with increased numbers of hydrophobic tumor antigens and a better response to immune checkpoint inhibitors independent of mutational load. Taken together, our findings demonstrate that UCD is a common feature of tumors that profoundly affects carcinogenesis, mutagenesis, and immunotherapy response.
Assuntos
Genômica , Metabolômica , Neoplasias/patologia , Ureia/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Animais , Aspartato Carbamoiltransferase/genética , Aspartato Carbamoiltransferase/metabolismo , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/genética , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/metabolismo , Linhagem Celular Tumoral , Di-Hidro-Orotase/genética , Di-Hidro-Orotase/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Proteínas de Transporte da Membrana Mitocondrial , Neoplasias/metabolismo , Ornitina Carbamoiltransferase/antagonistas & inibidores , Ornitina Carbamoiltransferase/genética , Ornitina Carbamoiltransferase/metabolismo , Fosforilação/efeitos dos fármacos , Pirimidinas/biossíntese , Pirimidinas/química , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismoRESUMO
Genome instability, defined as higher than normal rates of mutation, is a double-edged sword. As a source of genetic diversity and natural selection, mutations are beneficial for evolution. On the other hand, genomic instability can have catastrophic consequences for age-related diseases such as cancer. Mutations arise either from inactivation of DNA repair pathways or in a repair-competent background due to genotoxic stress from celluar processes such as transcription and replication that overwhelm high-fidelity DNA repair. Here, we review recent studies that shed light on endogenous sources of mutation and epigenomic features that promote genomic instability during cancer evolution.
Assuntos
Dano ao DNA , Instabilidade Genômica , Neoplasias/genética , Cromatina/química , Reparo do DNA , Replicação do DNA , Humanos , Mutação , Ativação TranscricionalRESUMO
Life inside ant colonies is orchestrated with diverse pheromones, but it is not clear how ants perceive these social signals. It has been proposed that pheromone perception in ants evolved via expansions in the numbers of odorant receptors (ORs) and antennal lobe glomeruli. Here, we generate the first mutant lines in the clonal raider ant, Ooceraea biroi, by disrupting orco, a gene required for the function of all ORs. We find that orco mutants exhibit severe deficiencies in social behavior and fitness, suggesting they are unable to perceive pheromones. Surprisingly, unlike in Drosophila melanogaster, orco mutant ants also lack most of the â¼500 antennal lobe glomeruli found in wild-type ants. These results illustrate that ORs are essential for ant social organization and raise the possibility that, similar to mammals, receptor function is required for the development and/or maintenance of the highly complex olfactory processing areas in the ant brain. VIDEO ABSTRACT.
Assuntos
Formigas/genética , Formigas/fisiologia , Proteínas de Insetos/metabolismo , Receptores Odorantes/metabolismo , Animais , Antenas de Artrópodes/citologia , Antenas de Artrópodes/fisiologia , Proteínas de Insetos/genética , Mutagênese , Mutação , Odorantes , Receptores Odorantes/genética , Comportamento SocialRESUMO
Genomes undergo different types of sporadic alterations, including DNA damage, point mutations, and genome rearrangements, that constitute the basis for evolution. However, these changes may occur at high levels as a result of cell pathology and trigger genome instability, a hallmark of cancer and a number of genetic diseases. In the last two decades, evidence has accumulated that transcription constitutes an important natural source of DNA metabolic errors that can compromise the integrity of the genome. Transcription can create the conditions for high levels of mutations and recombination by its ability to open the DNA structure and remodel chromatin, making it more accessible to DNA insulting agents, and by its ability to become a barrier to DNA replication. Here we review the molecular basis of such events from a mechanistic perspective with particular emphasis on the role of transcription as a genome instability determinant.
Assuntos
Reparo do DNA , Instabilidade Genômica , Mutagênese , Neoplasias/genética , Doenças Neurodegenerativas/genética , Transcrição Gênica , Montagem e Desmontagem da Cromatina , DNA/genética , DNA/metabolismo , Quebras de DNA de Cadeia Simples , Replicação do DNA , Genoma Humano , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Conformação de Ácido Nucleico , Recombinação GenéticaRESUMO
Antibiotic resistance is a global health threat and often results from new mutations. Antibiotics can induce mutations via mechanisms activated by stress responses, which both reveal environmental cues of mutagenesis and are weak links in mutagenesis networks. Network inhibition could slow the evolution of resistance during antibiotic therapies. Despite its pivotal importance, few identities and fewer functions of stress responses in mutagenesis are clear. Here, we identify the Escherichia coli stringent starvation response in fluoroquinolone-antibiotic ciprofloxacin-induced mutagenesis. Binding of response-activator ppGpp to RNA polymerase (RNAP) at two sites leads to an antibiotic-induced mutable gambler-cell subpopulation. Each activates a stress response required for mutagenic DNA-break repair: surprisingly, ppGpp-site-1-RNAP triggers the DNA-damage response, and ppGpp-site-2-RNAP induces σS-response activity. We propose that RNAP regulates DNA-damage processing in transcribed regions. The data demonstrate a critical node in ciprofloxacin-induced mutagenesis, imply RNAP-regulation of DNA-break repair, and identify promising targets for resistance-resisting drugs.
Assuntos
Proteínas de Escherichia coli , Proteínas de Escherichia coli/metabolismo , Guanosina Tetrafosfato/metabolismo , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Ciprofloxacina/farmacologia , DNA/metabolismo , RNA/metabolismo , Regulação Bacteriana da Expressão GênicaRESUMO
Germline mutations in the BRCA genes are associated with a higher risk of carcinogenesis, which is linked to an increased mutation rate and loss of the second unaffected BRCA allele (loss of heterozygosity, LOH). However, the mechanisms triggering mutagenesis are not clearly understood. The BRCA genes contain high numbers of repetitive DNA sequences. We detected replication forks stalling, DNA breaks, and deletions at these sites in haploinsufficient BRCA cells, thus identifying the BRCA genes as fragile sites. Next, we found that stalled forks are repaired by error-prone pathways, such as microhomology-mediated break-induced replication (MMBIR) in haploinsufficient BRCA1 breast epithelial cells. We detected MMBIR mutations in BRCA1 tumor cells and noticed deletions-insertions (>50 bp) at the BRCA1 genes in BRCA1 patients. Altogether, these results suggest that under stress, error-prone repair of stalled forks is upregulated and induces mutations, including complex genomic rearrangements at the BRCA genes (LOH), in haploinsufficient BRCA1 cells.
Assuntos
Proteína BRCA1 , Replicação do DNA , Humanos , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Reparo do DNA , Mutagênese , Genes BRCA1 , Perda de Heterozigosidade , Proteína BRCA2/genética , Proteína BRCA2/metabolismoRESUMO
Genomic DNA is continuously challenged by endogenous and exogenous sources of damage. The resulting lesions may act as physical blocks to DNA replication, necessitating repair mechanisms to be intrinsically coupled to the DNA replisome machinery. DNA damage tolerance (DDT) is comprised of translesion synthesis (TLS) and template switch (TS) repair processes that allow the replisome to bypass of bulky DNA lesions and complete DNA replication. How the replisome orchestrates which DDT repair mechanism becomes active at replication blocks has remained enigmatic. In this issue of Genes & Development, Dolce and colleagues (pp. 167-179) report that parental histone deposition by replisome components Ctf4 and Dpb3/4 promotes TS while suppressing error-prone TLS. Deletion of Dpb3/4 restored resistance to DNA-damaging agents in ctf4Δ cells at the expense of synergistic increases in mutagenesis due to elevated TLS. These findings illustrate the importance of replisome-directed chromatin maintenance to genome integrity and the response to DNA-damaging anticancer therapeutics.
Assuntos
Dano ao DNA , DNA , Dano ao DNA/genética , Reparo do DNA/genética , Replicação do DNARESUMO
Ctf4 is a conserved replisome component with multiple roles in DNA metabolism. To investigate connections between Ctf4-mediated processes involved in drug resistance, we conducted a suppressor screen of ctf4Δ sensitivity to the methylating agent MMS. We uncovered that mutations in Dpb3 and Dpb4 components of polymerase ε result in the development of drug resistance in ctf4Δ via their histone-binding function. Alleviated sensitivity to MMS of the double mutants was not associated with rescue of ctf4Δ defects in sister chromatid cohesion, replication fork architecture, or template switching, which ensures error-free replication in the presence of genotoxic stress. Strikingly, the improved viability depended on translesion synthesis (TLS) polymerase-mediated mutagenesis, which was drastically increased in ctf4 dpb3 double mutants. Importantly, mutations in Mcm2-Ctf4-Polα and Dpb3-Dpb4 axes of parental (H3-H4)2 deposition on lagging and leading strands invariably resulted in reduced error-free DNA damage tolerance through gap filling by template switch recombination. Overall, we uncovered a chromatin-based drug resistance mechanism in which defects in parental histone transfer after replication fork passage impair error-free recombination bypass and lead to up-regulation of TLS-mediated mutagenesis and drug resistance.
Assuntos
Histonas , Proteínas de Saccharomyces cerevisiae , Dano ao DNA/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Resistência a Medicamentos , Histonas/genética , Histonas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Microglia, the resident macrophages of the brain parenchyma, are key players in central nervous system (CNS) development, homeostasis, and disorders. Distinct brain pathologies seem associated with discrete microglia activation modules. How microglia regain quiescence following challenges remains less understood. Here, we explored the role of the interleukin-10 (IL-10) axis in restoring murine microglia homeostasis following a peripheral endotoxin challenge. Specifically, we show that lipopolysaccharide (LPS)-challenged mice harboring IL-10 receptor-deficient microglia displayed neuronal impairment and succumbed to fatal sickness. Addition of a microglial tumor necrosis factor (TNF) deficiency rescued these animals, suggesting a microglia-based circuit driving pathology. Single cell transcriptome analysis revealed various IL-10 producing immune cells in the CNS, including most prominently Ly49D+ NK cells and neutrophils, but not microglia. Collectively, we define kinetics of the microglia response to peripheral endotoxin challenge, including their activation and robust silencing, and highlight the critical role of non-microglial IL-10 in preventing deleterious microglia hyperactivation.
Assuntos
Endotoxinas/imunologia , Interleucina-10/metabolismo , Microglia/imunologia , Microglia/metabolismo , Animais , Biomarcadores , Encéfalo/imunologia , Encéfalo/metabolismo , Encéfalo/patologia , Células Cultivadas , Imunofenotipagem , Interleucina-10/genética , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Lipopolissacarídeos/imunologia , Ativação de Macrófagos , Macrófagos/imunologia , Macrófagos/metabolismo , CamundongosRESUMO
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.