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

Life Sci Alliance. 2023 Nov 2;7(1):e202302045. doi: 10.26508/lsa.202302045. Print 2024 Jan.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems / genetics
  • Cell Line
  • Cell Membrane / metabolism
  • Cystic Fibrosis Transmembrane Conductance Regulator* / genetics
  • Cystic Fibrosis Transmembrane Conductance Regulator* / metabolism
  • Cystic Fibrosis* / genetics
  • Cystic Fibrosis* / metabolism
  • Humans


  • Cystic Fibrosis Transmembrane Conductance Regulator
  • CFTR protein, human