RESUMO
CRISPR-based transcriptional activation is a powerful tool for functional gene interrogation; however, delivery difficulties have limited its applications in vivo. Here, we created a mouse model expressing all components of the CRISPR-Cas9 guide RNA-directed Synergistic Activation Mediator (SAM) from a single transcript that is capable of activating target genes in a tissue-specific manner. We optimized Lipid Nanoparticles and Adeno-Associated Virus guide RNA delivery approaches to achieve expression modulation of one or more genes in vivo. We utilized the SAM mouse model to generate a hypercholesteremia disease state that we could bidirectionally modulate with various guide RNAs. Additionally, we applied SAM to optimize gene expression in a humanized Transthyretin mouse model to recapitulate human expression levels. These results demonstrate that the SAM gene activation platform can facilitate in vivo research and drug discovery.
Assuntos
Sistemas CRISPR-Cas/genética , Hipercolesterolemia/genética , Lipossomos/farmacologia , Pré-Albumina/metabolismo , Ativação Transcricional/genética , Animais , Linhagem Celular , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Engenharia Genética/métodos , Células HEK293 , Humanos , Hipercolesterolemia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nanopartículas , Pré-Albumina/genética , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
The distribution of sense and antisense strand DNA mutations on transcribed duplex DNA contributes to the development of immune and neural systems along with the progression of cancer. Because developmentally matured B cells undergo biologically programmed strand-specific DNA mutagenesis at focal DNA/RNA hybrid structures, they make a convenient system to investigate strand-specific mutagenesis mechanisms. We demonstrate that the sense and antisense strand DNA mutagenesis at the immunoglobulin heavy chain locus and some other regions of the B cell genome depends upon localized RNA processing protein complex formation in the nucleus. Both the physical proximity and coupled activities of RNA helicase Mtr4 (and senataxin) with the noncoding RNA processing function of RNA exosome determine the strand-specific distribution of DNA mutations. Our study suggests that strand-specific DNA mutagenesis-associated mechanisms will play major roles in other undiscovered aspects of organismic development.
Assuntos
Linfócitos B/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Mutação , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Núcleo Celular/metabolismo , DNA Helicases/metabolismo , Exorribonucleases/genética , Instabilidade Genômica , Cadeias Pesadas de Imunoglobulinas/genética , Camundongos , Enzimas Multifuncionais , Proteínas Nucleares/genética , RNA Helicases , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/genéticaRESUMO
Regulatory B cells (Breg) have immune suppressive functions in various autoimmune/inflammation models and diseases and are found to be enriched in diverse B cell subsets. The lack of a unique marker or set of markers efficiently identifying Breg cells impedes detailed investigation into their origin, development, and immunological roles. Here, we perform transcriptome analysis of IL-10-expressing B cells to identify key regulators for Breg biogenesis and function and identify CD9, a tetraspanin-family transmembrane protein, as a key surface marker for most mouse IL-10(+) B cells and their progenitors. CD9 plays a role in the suppressive function of IL-10(+) B cells in ex vivo T cell proliferation assays through a mechanism that is dependent upon B/T cell interactions. CD9(+) B cells also demonstrate inhibition of Th1-mediated contact hypersensitivity in an in vivo model system. Taken together, our findings implicate CD9 in the immunosuppressive activity of regulatory B cells.