RT Journal Article
SR Electronic
T1 CRISPR/Cas9 bioluminescence-based assay for monitoring CFTR trafficking to the plasma membrane
JF Life Science Alliance
JO Life Sci. Alliance
FD Life Science Alliance LLC
SP e202302045
DO 10.26508/lsa.202302045
VO 7
IS 1
A1 Ondra, Martin
A1 Lenart, Lukas
A1 Centorame, Amanda
A1 Dumut, Daciana C
A1 He, Alexander
A1 Zaidi, Syeda Sadaf Zehra
A1 Hanrahan, John W
A1 De Sanctis, Juan Bautista
A1 Radzioch, Danuta
A1 Hajduch, Marian
YR 2024
UL http://www.life-science-alliance.org/content/7/1/e202302045.abstract
AB 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.