RESUMEN
Elucidation of gene function by reverse genetics in animal models frequently is complicated by the functional redundancy of homologous genes. This obstacle often is compounded by the tight clustering of homologous genes, which precludes the generation of multigene-deficient animals through standard interbreeding of single-deficient animals. Here, we describe an iterative, multiplexed CRISPR-based approach for simultaneous gene editing in the complex seven-member human airway trypsin-like protease/differentially expressed in a squamous cell carcinoma (HAT/DESC) cluster of membrane-anchored serine proteases. Through four cycles of targeting, we generated a library of 18 unique congenic mouse strains lacking combinations of HAT/DESC proteases, including a mouse strain deficient in all seven proteases. Using this library, we demonstrate that HAT/DESC proteases are dispensable for term development, postnatal health, and fertility and that the recently described function of the HAT-like 4 protease in epidermal barrier formation is unique among all HAT/DESC proteases. The study demonstrates the potential of iterative, multiplexed CRISPR-mediated gene editing for functional analysis of multigene clusters, and it provides a large array of new congenic mouse strains for the study of HAT/DESC proteases in physiological and in pathophysiological processes.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica/métodos , Familia de Multigenes , Serina Endopeptidasas/genética , Animales , Desarrollo Embrionario/genética , Epidermis/crecimiento & desarrollo , Epidermis/metabolismo , Femenino , Fertilidad/genética , Células HEK293 , Humanos , Masculino , Ratones , Serina Endopeptidasas/metabolismoRESUMEN
BACKGROUND: Matriptase is a membrane serine protease essential for epithelial development, homeostasis, and regeneration, as well as a central orchestrator of pathogenic pericellular signaling in the context of inflammatory and proliferative diseases. Matriptase is an unusual protease in that its zymogen displays measurable enzymatic activity. RESULTS: Here, we used gain and loss of function genetics to investigate the possible biological functions of zymogen matriptase. Unexpectedly, transgenic mice mis-expressing a zymogen-locked version of matriptase in the epidermis displayed pathologies previously reported for transgenic mice mis-expressing wildtype epidermal matriptase. Equally surprising, mice engineered to express only zymogen-locked endogenous matriptase, unlike matriptase null mice, were viable, developed epithelial barrier function, and regenerated the injured epithelium. Compatible with these observations, wildtype and zymogen-locked matriptase were equipotent activators of PAR-2 inflammatory signaling. CONCLUSION: The study demonstrates that the matriptase zymogen is biologically active and is capable of executing developmental and homeostatic functions of the protease.
Asunto(s)
Precursores Enzimáticos/metabolismo , Epitelio/crecimiento & desarrollo , Homeostasis/genética , Regeneración/genética , Serina Endopeptidasas/metabolismo , Animales , Precursores Enzimáticos/genética , Epitelio/metabolismo , Femenino , Mutación con Ganancia de Función , Expresión Génica , Mutación con Pérdida de Función , Masculino , Ratones Transgénicos , Serina Endopeptidasas/genéticaRESUMEN
Nitric oxide (NOâ¢) is a ubiquitous molecular mediator in biology. Many signalling actions of NO⢠generated by mammalian NO⢠synthase (NOS) result from targeting of the haem moiety of soluble guanylate cyclase. Some pathogenic and environmental bacteria also produce a NOS that is evolutionary related to the mammalian enzymes, but a bacterial haem-containing receptor for endogenous enzymatically generated NO⢠has not been identified previously. Here, we show that NOS of the human pathogen Staphylococcus aureus, in concert with an NOâ¢-metabolizing flavohaemoprotein, regulates electron transfer by targeting haem-containing cytochrome oxidases under microaerobic conditions to maintain membrane bioenergetics. This process is essential for staphylococcal nasal colonization and resistance to the membrane-targeting antibiotic daptomycin and demonstrates the conservation of NOS-derived NOâ¢-haem receptor signalling between bacteria and mammals.