S Gago, DW Denning, P Bowyer


Aspergillosis comprises a wide spectrum of fungal diseases with clinical manifestations that range from disseminated, chronic or colonization. One form of clinical disease may evolve into another over time depending on the immune status of the host but it is not known why all patients with comparable immune impairment do not suffer from disease. The aim of this study is to re-create genetic variants associated with susceptibility to allergic bronchopulmonary aspergillosis (ABPA) and chronic pulmonary aspergillosis (CPA) in human bronchial epithelial cells and, to study their importance in susceptibility to Aspergillus exposure.
Variants involved in susceptibility to ABPA and CPA were identified in a large exome sequencing study. The four most important genetic variants in three genes involved in epithelium maintenance, were recreated in 16HBE bronchial epithelial cells by using CRISPR-Cas9. To construct CRISPR-Cas9 plasmids targeting human genes, sense and antisense oligonucleotides were designed and cloned into an all-in-one vector containing OFP as a reporter. Forty-eight hours after transfection, cells were analysed and sorted by FACS and cell cultures propagated for 3 weeks. Sequence analysis was performed to confirm gene modification. Changes in the morphology of the epithelial monolayer and in its transmembrane resistance were determined. Moreover, CRISPR cell lines were challenged with Aspergillus fumigatus spores (CEA10) and incubated at 37C for 4h and changes in the epithelial integrity were microscopically observed.
We have standardized a CRISPR-Cas9 genome-editing system to introduce genetic variants associated with ABPA and CPA in human bronchial epithelial cell lines. The efficiency of the transfection was higher than 70% and the re-created cells could be propagated in culture. Genome editing was confirmed by sequencing a 500bp of the target region. Striking differences in the structure of the epithelial monolayer in CRISPR cell lines were observed when comparing to 16HBE cells suggesting a role for these genes in epithelium maintenance. Herein, the re-created epithelial cells showed a 30-40% reduction in transepithelial resistance compared with non-transfected cells. Moreover, the CRISPR cell lines were more susceptible to Aspergillus exposure and epithelial desquamation 4 hours after infection with multiplicity of infection ranging from 1-0.1.
Our study reveals novel insight into genetic susceptibility to fungal disease and provides proof-of-concept for the generation on in-vitro models of aspergillosis via CRISPR-Cas9 mediated gene targeting. More studies are needed to functionally confirm the role of these mutations in the different clinical forms of aspergillosis.


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7th Advances Against Aspergillosis conference