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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...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Life Science Alliance LLC
2023
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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 |
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author | 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 |
author_facet | 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 |
author_sort | Ondra, Martin |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-10622324 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Life Science Alliance LLC |
record_format | MEDLINE/PubMed |
spelling | pubmed-106223242023-11-04 CRISPR/Cas9 bioluminescence-based assay for monitoring CFTR trafficking to the plasma membrane 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 Life Sci Alliance Methods 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. Life Science Alliance LLC 2023-11-02 /pmc/articles/PMC10622324/ /pubmed/37918963 http://dx.doi.org/10.26508/lsa.202302045 Text en © 2023 Ondra et al. https://creativecommons.org/licenses/by/4.0/This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Methods 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 CRISPR/Cas9 bioluminescence-based assay for monitoring CFTR trafficking to the plasma membrane |
title | CRISPR/Cas9 bioluminescence-based assay for monitoring CFTR trafficking to the plasma membrane |
title_full | CRISPR/Cas9 bioluminescence-based assay for monitoring CFTR trafficking to the plasma membrane |
title_fullStr | CRISPR/Cas9 bioluminescence-based assay for monitoring CFTR trafficking to the plasma membrane |
title_full_unstemmed | CRISPR/Cas9 bioluminescence-based assay for monitoring CFTR trafficking to the plasma membrane |
title_short | CRISPR/Cas9 bioluminescence-based assay for monitoring CFTR trafficking to the plasma membrane |
title_sort | crispr/cas9 bioluminescence-based assay for monitoring cftr trafficking to the plasma membrane |
topic | Methods |
url | 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 |
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