CFTR Inactivation by Lentiviral Vector-mediated RNA Interference and CRISPR-Cas9 Genome Editing in Human Airway Epithelial Cells
Tuan Huy Nguyen.
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 for various pathologies. More recently,
genome editing using CRISPR-Cas9 artificial endonuclease was a valuable addition to the toolbox of
gene inactivation. Methods: Calu-3 cells and primary HAECs were transduced with HIV-1-derived
lentiviral vectors (LVV) encoding small hairpin RNA (shRNA) sequence or CRISPR-Cas9 components
targeting CFTR alongside GFP. After sorting of GFP-positive cells, CFTR expression was measured by RT-qPCR
and Western blot in polarized or differentiated cells. CFTR channel function was assessed in Ussing chambers. Il-8 secretion,
proliferation and cell migration were also studied in transduced cells. Results: shRNA interference and CRISPRCas9
strategies efficiently decreased CFTR expression in Calu-3 cells. Strong CFTR knockdown was confirmed at the
functional level in CRISPR-Cas9-modified cells. CFTR-specific shRNA sequences did not reduce gene expression in primary
HAECs, whereas CRISPR-Cas9-mediated gene modification activity was correlated with a reduction of transepithelial
secretion and response to a CFTR inhibitor. CFTR inactivation in the CRISPR-Cas9-modified Calu-3 cells did not affect
migration and proliferation but slightly increased basal interleukin-8 secretion. Conclusion: We generated CFTRinactivated
cell lines and demonstrated that CRISPR-Cas9 vectorised in a single LVV efficiently promotes CFTR inactivation
in primary HAECs. These results provide a new protocol to engineer CF primary epithelia with their isogenic controls
and pave the way for manipulation of CFTR expression in these cultures.
Keywords: CFTR, CRISPR-Cas9, Cystic fibrosis, Lentiviral vector, Primary cells, RNA interference.
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