CFTR Inactivation by Lentiviral Vector-mediated RNA Interference and CRISPR-Cas9 Genome Editing in Human Airway Epithelial Cells
2015
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.
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