(668) CRISPR based cellular models with endogenous expression of HiBiT tagged wild type and mutant CFTR enables high-throughput biology studies and screening for new transporter modulators

Background: CRISPR/Cas9 technology is a powerful gene editing tool for the insertion of pathogenic mutations in WT-CFTR that cause cystic fibrosis. Its application can be useful in preparing cellular models suitable for high-throughput screening (HTS). Moreover, it can be used to knock-in specific DNA sequences, coding for peptide tags, useful in the visualization and quantification of CFTR levels without interfering with its endogenous expression. Here we present a novel approach for the introduction of pathogenic mutations into WT-CFTR with HiBiT tag [1], suitable for HTS, aiming at the discovery of a novel combination of CFTR correctors and modulators that work in the presence of bacterial and fungal infection.

Methods: Firstly, HiBiT tag was inserted into the 4^th extracellular loop of WT-CFTR (human bronchial epithelial cells 16HBE14o-) by CRISPR/Cas9 and its function was validated. Secondly, we developed a workflow for the insertion of CFTR pathogenic mutation into WT-CFTR-HiBiT, and a screening pipeline for the discovery of novel CFTR correctors and modifiers compatible with our HTS core facility. In parallel, the identified modulators/correctors will be subjected to cytotoxicity screening.

Results: The workflow for the insertion of CFTR pathogenic mutations into WT-CFTR-HiBiT with endogenous expression was established. Our screening pipeline demonstrates high reproducibility and robustness. We implemented our novel screening tool in our HTS core facility. The selected combination of lead compounds will be biologically and pharmacologically compared to the effect of elexacaftor/tezacaftor/ivacaftor (triple combo) combination.

Conclusions: A novel high-throughput cell biology tool that allows for luminescent detection of total and cell-surface WT-CFTR expression driven by its endogenous promoter has been successfully validated. The approach for insertion of CFTR pathogenic mutation into WT-CFTR-HiBiT with endogenous expression and a powerful high-throughput screening tool has been established. This will allow to select new compounds and/or combination of modulators that work even under the conditions in which triple combo is not working efficiently.

Acknowledgements: This work was supported by IGA_LF_2022_012, the Czech Ministry of Education, Youth and Sports (EATRIS-CZ, LM2018133), Project ENOCH CZ.02.1.01/0.0/0.0/16_019/0000868, PSVT2B grant from MESI and Laurent Pharmaceuticals Inc.

References: [1] Schwinn MK, Machleidt T, Zimmerman K, et al. CRISPR-mediated tagging of endogenous proteins with a luminescent peptide. ACS Chem Biol 2018;13:467-74. https://doi.org/10.1021/acschembio.7b00549.