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

Autor:	Ondra M; https://ror.org/04qxnmv42 Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic.; https://ror.org/04qxnmv42 Czech Advanced Technology and Research Institute, Palacky University, Olomouc, Czech Republic., Lenart L; https://ror.org/04qxnmv42 Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic., Centorame A; https://ror.org/01pxwe438 Faculty of Medicine and Health Sciences, McGill University, Montreal, Canada.; RI-MUHC, Montreal, Canada., Dumut DC; https://ror.org/01pxwe438 Faculty of Medicine and Health Sciences, McGill University, Montreal, Canada.; RI-MUHC, Montreal, Canada., He A; https://ror.org/01pxwe438 Physiology, McGill University, Montreal, Canada., Zaidi SSZ; https://ror.org/01pxwe438 Physiology, McGill University, Montreal, Canada., Hanrahan JW; RI-MUHC, Montreal, Canada.; https://ror.org/01pxwe438 Physiology, McGill University, Montreal, Canada., De Sanctis JB; https://ror.org/04qxnmv42 Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic., Radzioch D; https://ror.org/04qxnmv42 Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic danuta.radzioch@mcgill.ca.; https://ror.org/01pxwe438 Faculty of Medicine and Health Sciences, McGill University, Montreal, Canada.; RI-MUHC, Montreal, Canada., Hajduch M; https://ror.org/04qxnmv42 Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic marian.hajduch@upol.cz.; https://ror.org/04qxnmv42 Czech Advanced Technology and Research Institute, Palacky University, Olomouc, Czech Republic.; Laboratory of Experimental Medicine, Institute of Molecular and Translational Medicine, University Hospital Olomouc, Olomouc, Czech Republic.
Jazyk:	angličtina
Zdroj:	Life science alliance [Life Sci Alliance] 2023 Nov 02; Vol. 7 (1). Date of Electronic Publication: 2023 Nov 02 (Print Publication: 2024).
DOI:	10.26508/lsa.202302045
Abstrakt:	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. (© 2023 Ondra et al.)
Databáze:	MEDLINE
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