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CRISPR/Cas9 bioluminescence-based assay for monitoring CFTR trafficking to the plasma membrane

CFTR is a membrane protein that functions as an ion channel. Mutations that disrupt its biosynthesis, trafficking or function cause cystic fibrosis (CF). Here, we present a novel in vitro model system prepared using CRISPR/Cas9 genome editing with endogenously expressed WT-CFTR tagged with a HiBiT p...

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Detalles Bibliográficos
Autores principales: Ondra, Martin, Lenart, Lukas, Centorame, Amanda, Dumut, Daciana C, He, Alexander, Zaidi, Syeda Sadaf Zehra, Hanrahan, John W, De Sanctis, Juan Bautista, Radzioch, Danuta, Hajduch, Marian
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Life Science Alliance LLC 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10622324/
https://www.ncbi.nlm.nih.gov/pubmed/37918963
http://dx.doi.org/10.26508/lsa.202302045
Descripción
Sumario:CFTR is a membrane protein that functions as an ion channel. Mutations that disrupt its biosynthesis, trafficking or function cause cystic fibrosis (CF). Here, we present a novel in vitro model system prepared using CRISPR/Cas9 genome editing with endogenously expressed WT-CFTR tagged with a HiBiT peptide. To enable the detection of CFTR in the plasma membrane of live cells, we inserted the HiBiT tag in the fourth extracellular loop of WT-CFTR. The 11-amino acid HiBiT tag binds with high affinity to a large inactive subunit (LgBiT), generating a reporter luciferase with bright luminescence. Nine homozygous clones with the HiBiT knock-in were identified from the 182 screened clones; two were genetically and functionally validated. In summary, this work describes the preparation and validation of a novel reporter cell line with the potential to be used as an ultimate building block for developing unique cellular CF models by CRISPR-mediated insertion of CF-causing mutations.