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.

Czech Advanced Technology and Research Institute Palacky University Olomouc Czech Republic

Faculty of Medicine and Health Sciences McGill University Montreal Canada

Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacky University Olomouc Czech Republic

Laboratory of Experimental Medicine Institute of Molecular and Translational Medicine University Hospital Olomouc Olomouc Czech Republic

Physiology McGill University Montreal Canada

RI MUHC Montreal Canada

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Ameen N, Silvis M, Bradbury NA (2007) Endocytic trafficking of CFTR in health and disease. J Cyst Fibros 6: 1–14. 10.1016/j.jcf.2006.09.002 PubMed DOI PMC

Boucher RC (2007) Airway surface dehydration in cystic fibrosis: Pathogenesis and therapy. Annu Rev Med 58: 157–170. 10.1146/annurev.med.58.071905.105316 PubMed DOI

Boyle MP, De Boeck K (2013) A new era in the treatment of cystic fibrosis: Correction of the underlying CFTR defect. Lancet Respir Med 1: 158–163. 10.1016/S2213-2600(12)70057-7 PubMed DOI

Brinkman EK, Chen T, Amendola M, van Steensel B (2014) Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res 42: e168. 10.1093/nar/gku936 PubMed DOI PMC

Cantin AM, Hartl D, Konstan MW, Chmiel JF (2015) Inflammation in cystic fibrosis lung disease: Pathogenesis and therapy. J Cyst Fibros 14: 419–430. 10.1016/j.jcf.2015.03.003 PubMed DOI

Carlile GW, Robert R, Zhang D, Teske KA, Luo Y, Hanrahan JW, Thomas DY (2007) Correctors of protein trafficking defects identified by a novel high-throughput screening assay. ChemBioChem 8: 1012–1020. 10.1002/cbic.200700027 PubMed DOI

Chang X, Mengos A, Hou Y, Cui L, Jensen TJ, Aleksandrov A, Riordan JR, Gentzsch M (2008) Role of N-linked oligosaccharides in the biosynthetic processing of the cystic fibrosis membrane conductance regulator. J Cell Sci 121: 2814–2823. 10.1242/jcs.028951 PubMed DOI PMC

Cheng SH, Gregory RJ, Marshall J, Paul S, Souza DW, White GA, O’Riordan CR, Smith AE (1990) Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell 63: 827–834. 10.1016/0092-8674(90)90148-8 PubMed DOI

Clancy JP, Cotton CU, Donaldson SH, Solomon GM, VanDevanter DR, Boyle MP, Gentzsch M, Nick JA, Illek B, Wallenburg JC, et al. (2019) CFTR modulator theratyping: Current status, gaps and future directions. J Cyst Fibros 18: 22–34. 10.1016/j.jcf.2018.05.004 PubMed DOI PMC

Coakley RD, Grubb BR, Paradiso AM, Gatzy JT, Johnson LG, Kreda SM, O’Neal WK, Boucher RC (2003) Abnormal surface liquid pH regulation by cultured cystic fibrosis bronchial epithelium. Proc Natl Acad Sci U S A 100: 16083–16088. 10.1073/pnas.2634339100 PubMed DOI PMC

Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, et al. (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339: 819–823. 10.1126/science.1231143 PubMed DOI PMC

Cozens AL, Yezzi MJ, Kunzelmann K, Ohrui T, Chin L, Eng K, Finkbeiner WE, Widdicombe JH, Gruenert DC (1994) CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells. Am J Respir Cell Mol Biol 10: 38–47. 10.1165/ajrcmb.10.1.7507342 PubMed DOI

Cutting GR (2010) Modifier genes in mendelian disorders: The example of cystic fibrosis. Ann N Y Acad Sci 1214: 57–69. 10.1111/j.1749-6632.2010.05879.x PubMed DOI PMC

Cutting GR (2015) Cystic fibrosis genetics: From molecular understanding to clinical application. Nat Rev Genet 16: 45–56. 10.1038/nrg3849 PubMed DOI PMC

Dean M, Rzhetsky A, Allikmets R (2001) The human ATP-binding cassette (ABC) transporter superfamily. Genome Res 11: 1156–1166. 10.1101/gr.184901 PubMed DOI

Della Sala A, Prono G, Hirsch E, Ghigo A (2021) Role of protein kinase A-mediated phosphorylation in CFTR channel activity regulation. Front Physiol 12: 690247. 10.3389/fphys.2021.690247 PubMed DOI PMC

Dixon AS, Schwinn MK, Hall MP, Zimmerman K, Otto P, Lubben TH, Butler BL, Binkowski BF, Machleidt T, Kirkland TA, et al. (2016) NanoLuc complementation reporter optimized for accurate measurement of protein interactions in cells. ACS Chem Biol 11: 400–408. 10.1021/acschembio.5b00753 PubMed DOI

Donaldson JG, Finazzi D, Klausner RD (1992) Brefeldin A inhibits Golgi membrane-catalysed exchange of guanine nucleotide onto ARF protein. Nature 360: 350–352. 10.1038/360350a0 PubMed DOI

Dow LE (2015) Modeling disease in vivo with CRISPR/Cas9. Trends Mol Med 21: 609–621. 10.1016/j.molmed.2015.07.006 PubMed DOI PMC

Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411: 494–498. 10.1038/35078107 PubMed DOI

Farinha CM, Canato S (2017) From the endoplasmic reticulum to the plasma membrane: Mechanisms of CFTR folding and trafficking. Cell Mol Life Sci 74: 39–55. 10.1007/s00018-016-2387-7 PubMed DOI PMC

Galietta LJV, Haggie PM, Verkman AS (2001. a) Green fluorescent protein-based halide indicators with improved chloride and iodide affinities. FEBS Lett 499: 220–224. 10.1016/S0014-5793(01)02561-3 PubMed DOI

Galietta LV, Jayaraman S, Verkman AS (2001. b) Cell-based assay for high-throughput quantitative screening of CFTR chloride transport agonists. Am J Physiol Cell Physiol 281: C1734–C1742. 10.1152/ajpcell.2001.281.5.C1734 PubMed DOI

Gianotti A, Delpiano L, Caci E (2018) In vitro methods for the development and analysis of human primary airway epithelia. Front Pharmacol 9: 1176. 10.3389/fphar.2018.01176 PubMed DOI PMC

Gregory RJ, Cheng SH, Rich DP, Marshall J, Paul S, Hehir K, Ostedgaard L, Klinger KW, Welsh MJ, Smith AE (1990) Expression and characterization of the cystic fibrosis transmembrane conductance regulator. Nature 347: 382–386. 10.1038/347382a0 PubMed DOI

Guo J, Garratt A, Hill A (2022) Worldwide rates of diagnosis and effective treatment for cystic fibrosis. J Cyst Fibros 21: 456–462. 10.1016/j.jcf.2022.01.009 PubMed DOI

Hall MP, Unch J, Binkowski BF, Valley MP, Butler BL, Wood MG, Otto P, Zimmerman K, Vidugiris G, Machleidt T, et al. (2012) Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol 7: 1848–1857. 10.1021/cb3002478 PubMed DOI PMC

Howard M, DuVall MD, Devor DC, Dong JY, Henze K, Frizzell RA (1995) Epitope tagging permits cell surface detection of functional CFTR. Am J Physiol 269: C1565–C1576. 10.1152/ajpcell.1995.269.6.C1565 PubMed DOI

Illek B, Maurisse R, Wahler L, Kunzelmann K, Fischer H, Gruenert DC (2008) Cl transport in complemented CF bronchial epithelial cells correlates with CFTR mRNA expression levels. Cell Physiol Biochem 22: 57–68. 10.1159/000149783 PubMed DOI PMC

Jayaraman S, Haggie P, Wachter RM, Remington SJ, Verkman AS (2000) Mechanism and cellular applications of a green fluorescent protein-based halide sensor. J Biol Chem 275: 6047–6050. 10.1074/jbc.275.9.6047 PubMed DOI

Jia Y, Mathews CJ, Hanrahan JW (1997) Phosphorylation by protein kinase C is required for acute activation of cystic fibrosis transmembrane conductance regulator by protein kinase A. J Biol Chem 272: 4978–4984. 10.1074/jbc.272.8.4978 PubMed DOI

Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 337: 816–821. 10.1126/science.1225829 PubMed DOI PMC

Kampmann M (2020) CRISPR-based functional genomics for neurological disease. Nat Rev Neurol 16: 465–480. 10.1038/s41582-020-0373-z PubMed DOI PMC

Kerem BS, Rommens JM, Buchanan JA, Markiewicz D, Cox TK, Chakravarti A, Buchwald M, Tsui LC (1989) Identification of the cystic fibrosis gene: Genetic analysis. Science 245: 1073–1080. 10.1126/science.2570460 PubMed DOI

Kim H, Kim JS (2014) A guide to genome engineering with programmable nucleases. Nat Rev Genet 15: 321–334. 10.1038/nrg3686 PubMed DOI

Konstantakos V, Nentidis A, Krithara A, Paliouras G (2022) CRISPR–Cas9 gRNA efficiency prediction: An overview of predictive tools and the role of deep learning. Nucleic Acids Res 50: 3616–3637. 10.1093/nar/gkac192 PubMed DOI PMC

Labun K, Montague TG, Krause M, Torres Cleuren YN, Tjeldnes H, Valen E (2019) CHOPCHOP v3: Expanding the CRISPR web toolbox beyond genome editing. Nucleic Acids Res 47: W171–W174. 10.1093/nar/gkz365 PubMed DOI PMC

Liu F, Zhang Z, Csanády L, Gadsby DC, Chen J (2017) Molecular structure of the human CFTR ion channel. Cell 169: 85–95.e8. 10.1016/j.cell.2017.02.024 PubMed DOI

Lopes-Pacheco M (2016) CFTR modulators: Shedding light on precision medicine for cystic fibrosis. Front Pharmacol 7: 275. 10.3389/fphar.2016.00275 PubMed DOI PMC

Lopes-Pacheco M (2020) CFTR modulators: The changing face of cystic fibrosis in the era of precision medicine. Front Pharmacol 10: 1662. 10.3389/fphar.2019.01662 PubMed DOI PMC

Ma T, Vetrivel L, Yang H, Pedemonte N, Zegarra-Moran O, Galietta LJV, Verkman AS (2002) High-affinity activators of cystic fibrosis transmembrane conductance regulator (CFTR) chloride conductance identified by high-throughput screening. J Biol Chem 277: 37235–37241. 10.1074/jbc.M205932200 PubMed DOI

Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 339: 823–826. 10.1126/science.1232033 PubMed DOI PMC

Mall M, Hipper A, Greger R, Kunzelmann K (1996) Wild type but not deltaF508 CFTR inhibits Na+ conductance when coexpressed in Xenopus oocytes. FEBS Lett 381: 47–52. 10.1016/0014-5793(96)00079-8 PubMed DOI

Meacham GC, Patterson C, Zhang W, Younger JM, Cyr DM (2001) The Hsc70 co-chaperone CHIP targets immature CFTR for proteasomal degradation. Nat Cell Biol 3: 100–105. 10.1038/35050509 PubMed DOI

Molina SA, Hunt WR (2017) Chapter 12 - cystic fibrosis: An overview of the past, present, and the future. In Lung Epithelial Biology in the Pathogenesis of Pulmonary Disease, Sidhaye VK, Koval M (eds), pp 219–249. Cambridge, MA: Academic Press.

Moriya H (2015) Quantitative nature of overexpression experiments. Mol Biol Cell 26: 3932–3939. 10.1091/mbc.E15-07-0512 PubMed DOI PMC

Moyer BD, Loffing J, Schwiebert EM, Loffing-Cueni D, Halpin PA, Karlson KH, Ismailov II, Guggino WB, Langford GM, Stanton BA (1998) Membrane trafficking of the cystic fibrosis gene product, cystic fibrosis transmembrane conductance regulator, tagged with green fluorescent protein in madin-darby canine kidney cells. J Biol Chem 273: 21759–21768. 10.1074/jbc.273.34.21759 PubMed DOI

Pedemonte N, Tomati V, Sondo E, Galietta LJV (2010) Influence of cell background on pharmacological rescue of mutant CFTR. Am J Physiol Cell Physiol 298: C866–C874. 10.1152/ajpcell.00404.2009 PubMed DOI

Pezzulo AA, Tang XX, Hoegger MJ, Abou Alaiwa MH, Ramachandran S, Moninger TO, Karp PH, Wohlford-Lenane CL, Haagsman HP, van Eijk M, et al. (2012) Reduced airway surface pH impairs bacterial killing in the porcine cystic fibrosis lung. Nature 487: 109–113. 10.1038/nature11130 PubMed DOI PMC

Poulsen JH, Fischer H, Illek B, Machen TE (1994) Bicarbonate conductance and pH regulatory capability of cystic fibrosis transmembrane conductance regulator. Proc Natl Acad Sci U S A 91: 5340–5344. 10.1073/pnas.91.12.5340 PubMed DOI PMC

Prelich G (2012) Gene overexpression: Uses, mechanisms, and interpretation. Genetics 190: 841–854. 10.1534/genetics.111.136911 PubMed DOI PMC

Ramalho AS, Boon M, Proesmans M, Vermeulen F, Carlon MS, Boeck KD (2022) Assays of CFTR function in vitro, ex vivo and in vivo. Int J Mol Sci 23: 1437. 10.3390/ijms23031437 PubMed DOI PMC

Riordan JR, Rommens JM, Kerem BS, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou JL, et al. (1989) Identification of the cystic fibrosis gene: Cloning and characterization of complementary DNA. Science 245: 1066–1073. 10.1126/science.2475911 PubMed DOI

Rommens JM, Iannuzzi MC, Kerem BS, Drumm ML, Melmer G, Dean M, Rozmahel R, Cole JL, Kennedy D, Hidaka N, et al. (1989) Identification of the cystic fibrosis gene: Chromosome walking and jumping. Science 245: 1059–1065. 10.1126/science.2772657 PubMed DOI

Rowe SM, Verkman AS (2013) Cystic fibrosis transmembrane regulator correctors and potentiators. Cold Spring Harb Perspect Med 3: a009761. 10.1101/cshperspect.a009761 PubMed DOI PMC

Ruan J, Hirai H, Yang D, Ma L, Hou X, Jiang H, Wei H, Rajagopalan C, Mou H, Wang G, et al. (2019) Efficient gene editing at major CFTR mutation loci. Mol Ther Nucleic Acids 16: 73–81. 10.1016/j.omtn.2019.02.006 PubMed DOI PMC

Schwinn MK, Machleidt T, Zimmerman K, Eggers CT, Dixon AS, Hurst R, Hall MP, Encell LP, Binkowski BF, Wood KV (2018) CRISPR-mediated tagging of endogenous proteins with a luminescent peptide. ACS Chem Biol 13: 467–474. 10.1021/acschembio.7b00549 PubMed DOI

Sheppard DN, Welsh MJ (1999) Structure and function of the CFTR chloride channel. Physiol Rev 79: S23–S45. 10.1152/physrev.1999.79.1.S23 PubMed DOI

Smith E, Giuliano KA, Shumate J, Baillargeon P, McEwan B, Cullen MD, Miller JP, Drew L, Scampavia L, Spicer TP (2017) A homogeneous cell-based halide-sensitive yellow fluorescence protein assay to identify modulators of the cystic fibrosis transmembrane conductance regulator ion channel. Assay Drug Dev Technol 15: 395–406. 10.1089/adt.2017.810 PubMed DOI

Sorum B, Töröcsik B, Csanády L (2017) Asymmetry of movements in CFTR's two ATP sites during pore opening serves their distinct functions. Elife 6: e29013. 10.7554/eLife.29013 PubMed DOI PMC

Stutts MJ, Canessa CM, Olsen JC, Hamrick M, Cohn JA, Rossier BC, Boucher RC (1995) CFTR as a cAMP-dependent regulator of sodium channels. Science 269: 847–850. 10.1126/science.7543698 PubMed DOI

Tabcharani JA, Chang XB, Riordan JR, Hanrahan JW (1991) Phosphorylation-regulated Cl- channel in CHO cells stably expressing the cystic fibrosis gene. Nature 352: 628–631. 10.1038/352628a0 PubMed DOI

The clinical and Functional TRanslation of CFTR (2023) CFTR2 database. Available at: https://cftr2.org.

Tsui LC, Dorfman R (2013) The cystic fibrosis gene: A molecular genetic perspective. Cold Spring Harb Perspect Med 3: a009472. 10.1101/cshperspect.a009472 PubMed DOI PMC

Turcios NL (2020) Cystic fibrosis lung disease: An overview. Respir Care 65: 233–251. 10.4187/respcare.06697 PubMed DOI

Veit G, Avramescu RG, Chiang AN, Houck SA, Cai Z, Peters KW, Hong JS, Pollard HB, Guggino WB, Balch WE, et al. (2016) From CFTR biology toward combinatorial pharmacotherapy: Expanded classification of cystic fibrosis mutations. Mol Biol Cell 27: 424–433. 10.1091/mbc.E14-04-0935 PubMed DOI PMC

Vergani P, Lockless SW, Nairn AC, Gadsby DC (2005) CFTR channel opening by ATP-driven tight dimerization of its nucleotide-binding domains. Nature 433: 876–880. 10.1038/nature03313 PubMed DOI PMC

Zhang Z, Liu F, Chen J (2017) Conformational changes of CFTR upon phosphorylation and ATP binding. Cell 170: 483–491.e8. 10.1016/j.cell.2017.06.041 PubMed DOI

Zhang C, Zhang G, Zhang Y, Lin X, Zhao X, Cui Q, Rong L, Du R (2023) Development of an HiBiT-tagged reporter H3N2 influenza A virus and its utility as an antiviral screening platform. J Med Virol 95: e28345. 10.1002/jmv.28345 PubMed DOI

Zielenski J (2000) Genotype and phenotype in cystic fibrosis. Respiration 67: 117–133. 10.1159/000029497 PubMed DOI