CFTR inactivation by lentiviral vector-mediated RNA interference and CRISPR-Cas9 genome editing in human airway epithelial cells.

摘要: Background: Polarized airway epithelial cell cultures modelling Cystic Fibrosis Transmembrane conductance Regulator ( CFTR ) defect are crucial for CF and biomedical research. RNA interference has proven its value to generate knockdown models various pathologies. More recently, genome editing using CRISPR-Cas9 artificial endonuclease was a valuable addition the toolbox of gene inactivation. Methods: Calu-3 cells primary HAECs were transduced with HIV-1-derived lentiviral vectors (LVV) encoding small hairpin (shRNA) sequence or components targeting alongside GFP. After sorting of GFP-positive cells, expression measured by RT-qPCR and Western blot in polarized differentiated cells. channel function assessed Ussing chambers. Il-8 secretion, proliferation migration also studied Results: shRNA interference CRISPRCas9 strategies efficiently decreased Strong confirmed at the functional level CRISPR-Cas9-modified -specific sequences did not reduce gene primary HAECs, whereas CRISPR-Cas9-mediated modification activity correlated reduction transepithelial secretion response inhibitor. inactivation affect migration proliferation but slightly increased basal interleukin-8 secretion. Conclusion: We generated CFTRinactivated cell lines demonstrated that vectorised single LVV promotes inactivation in HAECs. These results provide new protocol engineer epithelia their isogenic controls and pave way manipulation these cultures.