RESUMEN
The functional effect of a gene edit by designer nucleases depends on the DNA repair outcome at the targeted locus. While non-homologous end joining (NHEJ) repair results in various mutations, microhomology-mediated end joining (MMEJ) creates precise deletions based on the alignment of flanking microhomologies (µHs). Recently, the sequence context surrounding nuclease-induced double strand breaks (DSBs) has been shown to predict repair outcomes, for which µH plays an important role. Here, we survey naturally occurring human deletion variants and identify that 11 million or 57% are flanked by µHs, covering 88% of protein-coding genes. These biologically relevant mutations are candidates for precise creation in a template-free manner by MMEJ repair. Using CRISPR-Cas9 in human induced pluripotent stem cells (hiPSCs), we efficiently create pathogenic deletion mutations for demonstrable disease models with both gain- and loss-of-function phenotypes. We anticipate this dataset and gene editing strategy to enable functional genetic studies and drug screening.
Asunto(s)
Reparación del ADN por Unión de Extremidades/genética , Edición Génica/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Eliminación de Secuencia/genética , Secuencia de Bases , Sistemas CRISPR-Cas , Mutación con Ganancia de Función , Humanos , Mutación con Pérdida de FunciónRESUMEN
Proper niche architecture is critical for stem cell function, yet only few upstream regulators are known. Here, we report that the Hox transcription factor Abdominal-B (Abd-B), active in premeiotic spermatocytes of Drosophila testes, is essential for positioning the niche to the testis anterior by regulating integrin in neighboring somatic cyst cells. Abd-B also non-cell-autonomously controls critical features within the niche, including centrosome orientation and division rates of germline stem cells. By using genome-wide binding studies, we find that Abd-B mediates its effects on integrin localization by directly controlling at multiple levels the signaling activity of the Sev ligand Boss via its direct targets src42A and sec63, two genes involved in protein trafficking and recycling. Our data show that Abd-B, through local signaling between adjucent cell types, provides positional cues for integrin localization, which is critical for placement of the distant stem cell niche and stem cell activity.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteínas de Homeodominio/metabolismo , Nicho de Células Madre , Testículo/citología , Células Madre Adultas/citología , Células Madre Adultas/metabolismo , Células Madre Adultas/fisiología , Animales , División Celular , Centrosoma/metabolismo , Drosophila/citología , Drosophila/genética , Drosophila/crecimiento & desarrollo , Proteínas de Drosophila/genética , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Genoma de los Insectos , Proteínas de Homeodominio/genética , Integrinas/genética , Integrinas/metabolismo , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Péptidos/genética , Receptores de Péptidos/metabolismo , Espermatocitos/citología , Espermatocitos/metabolismo , Espermatocitos/fisiología , Testículo/crecimiento & desarrollo , Testículo/metabolismoRESUMEN
Heartbeat is required for normal development of the heart, and perturbation of intracardiac flow leads to morphological defects resembling congenital heart diseases. These observations implicate intracardiac haemodynamics in cardiogenesis, but the signalling cascades connecting physical forces, gene expression and morphogenesis are largely unknown. Here we use a zebrafish model to show that the microRNA, miR-21, is crucial for regulation of heart valve formation. Expression of miR-21 is rapidly switched on and off by blood flow. Vasoconstriction and increasing shear stress induce ectopic expression of miR-21 in the head vasculature and heart. Flow-dependent expression of mir-21 governs valvulogenesis by regulating the expression of the same targets as mouse/human miR-21 (sprouty, pdcd4, ptenb) and induces cell proliferation in the valve-forming endocardium at constrictions in the heart tube where shear stress is highest. We conclude that miR-21 is a central component of a flow-controlled mechanotransduction system in a physicogenetic regulatory loop.