RESUMO
In biological systems, spatial organization and function are interconnected. Here we present photoselective sequencing, a new method for genomic and epigenomic profiling within morphologically distinct regions. Starting with an intact biological specimen, photoselective sequencing uses targeted illumination to selectively unblock a photocaged fragment library, restricting the sequencing-based readout to microscopically identified spatial regions. We validate photoselective sequencing by measuring the chromatin accessibility profiles of fluorescently labeled cell types within the mouse brain and comparing with published data. Furthermore, by combining photoselective sequencing with a computational strategy for decomposing bulk accessibility profiles, we find that the oligodendrocyte-lineage-cell population is relatively enriched for oligodendrocyte-progenitor cells in the cortex versus the corpus callosum. Finally, we leverage photoselective sequencing at the subcellular scale to identify features of chromatin that are correlated with positioning at the nuclear periphery. These results collectively demonstrate that photoselective sequencing is a flexible and generalizable platform for exploring the interplay of spatial structures with genomic and epigenomic properties.
Assuntos
Cromatina , Genômica , Camundongos , Animais , Núcleo Celular , Epigenômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodosRESUMO
Duchenne muscular dystrophy (DMD) is typically caused by mutations that disrupt the DMD reading frame, but nonsense mutations in the 5' part of the gene induce utilization of an internal ribosomal entry site (IRES) in exon 5, driving expression of a highly functional N-truncated dystrophin. We have developed an AAV9 vector expressing U7 small nuclear RNAs targeting DMD exon 2 and have tested it in a mouse containing a duplication of exon 2, in which skipping of both exon 2 copies induces IRES-driven expression, and skipping of one copy leads to wild-type dystrophin expression. One-time intravascular injection either at postnatal days 0-1 or at 2 months results in efficient exon skipping and dystrophin expression, and significant protection from functional and pathologic deficits. Immunofluorescence quantification showed 33%-53% average dystrophin intensity and 55%-79% average dystrophin-positive fibers in mice treated in adulthood, with partial amelioration of DMD pathology and correction of DMD-associated alterations in gene expression. In mice treated neonatally, dystrophin immunofluorescence reached 49%-85% of normal intensity and 76%-99% dystrophin-positive fibers, with near-complete correction of dystrophic pathology, and these beneficial effects persisted for at least 6 months. Our results demonstrate the robustness, durability, and safety of exon 2 skipping using scAAV9.U7snRNA.ACCA, supporting its clinical use.
RESUMO
The uridine-rich 7 (U7) small nuclear RNA (snRNA) is a component of a small nuclear ribonucleoprotein (snRNP) complex. U7 snRNA naturally contains an antisense sequence that identifies histone premessenger RNAs (pre-mRNAs) and is involved in their 3' end processing. By altering this antisense sequence, researchers have turned U7 snRNA into a versatile tool for targeting pre-mRNAs and modifying splicing. Encapsulating a modified U7 snRNA into a viral vector such as adeno-associated virus (also referred as vectorized exon skipping/inclusion, or VES/VEI) enables the delivery of this highly efficacious splicing modulator into a range of cell lines, primary cells, and tissues. In addition, and in contrast to antisense oligonucleotides, viral delivery of U7 snRNA enables long-term expression of antisense sequences in the nucleus as part of a stable snRNP complex. As a result, VES/VEI has emerged as a promising therapeutic platform for treating a large variety of human diseases caused by errors in pre-mRNA splicing or its regulation. Here we provide an overview of U7 snRNA's natural function and its applications in gene therapy.
Assuntos
RNA Nuclear Pequeno , RNA , Terapia Genética , Vetores Genéticos , Humanos , Precursores de RNA , RNA Nuclear Pequeno/genéticaRESUMO
Single-cell RNA sequencing has revealed extensive molecular diversity in gene programs governing mammalian spermatogenesis but fails to delineate their dynamics in the native context of seminiferous tubules, the spatially confined functional units of spermatogenesis. Here, we use Slide-seq, a spatial transcriptomics technology, to generate an atlas that captures the spatial gene expression patterns at near-single-cell resolution in the mouse and human testis. Using Slide-seq data, we devise a computational framework that accurately localizes testicular cell types in individual seminiferous tubules. Unbiased analysis systematically identifies spatially patterned genes and gene programs. Combining Slide-seq with targeted in situ RNA sequencing, we demonstrate significant differences in the cellular compositions of spermatogonial microenvironment between mouse and human testes. Finally, a comparison of the spatial atlas generated from the wild-type and diabetic mouse testis reveals a disruption in the spatial cellular organization of seminiferous tubules as a potential mechanism of diabetes-induced male infertility.
Assuntos
Perfilação da Expressão Gênica , Espermatogênese/genética , Espermatogônias/metabolismo , Testículo/metabolismo , Transcriptoma , Algoritmos , Animais , Microambiente Celular , Bases de Dados Genéticas , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , RNA-Seq , Análise de Célula Única , Especificidade da Espécie , Espermatogônias/patologia , Testículo/patologia , Fatores de TempoRESUMO
Here we describe TRACE (T7 polymerase-driven continuous editing), a method that enables continuous, targeted mutagenesis in human cells using a cytidine deaminase fused to T7 RNA polymerase. TRACE induces high rates of mutagenesis over multiple cell generations in genes under the control of a T7 promoter integrated in the genome. We used TRACE in a MEK1 inhibitor-resistance screen, and identified functionally correlated mutations.