Crispr cas9 technology

Madin-Darby Canine Kidney epithelial (MDCK, American Type Culture collection, ATCC CCL-34), human embryonic kidney (293T; ATCC CRL-11268), human lung epithelial carcinoma (A549; ATCC CCL-185), and African green monkey kidney epithelial E6 (Vero E6, kindly provided by Prof. Luis Enjuanes, Centro Nacional de Biotecnología, CNB-CSIC, Spain) cells were grown at 37°C in air enriched with 5% CO₂ using Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (Gibco), and 50 μg/ml gentamicin (Gibco). A549 cells overexpressing human ACE-2 (hACE-2, A549-ACE-2), generated in our laboratory, were grown in the same media containing 2.5 μg/ml of blasticidin (ThermoFisher Scientific). Human HAP-1 near-haploid wild-type (WT) cells, derived from a human leukemia, and HAP-1 cells knock-out (KO) for IFI6 using the CRISPR/Cas9 technology were obtained from Horizon Discovery, Inc. These cells were grown at 37°C in air enriched with 5% CO₂ using Iscove´s Modified Dulbecco´s medium supplemented with 10% fetal bovine serum (Gibco), and 50 μg/ml gentamicin (Gibco). The non-tumorigenic human bronchial epithelial cell line BEAS-2B (CRL-9609), was obtained from the ATCC and grown at 37°C in air enriched with 5% CO using RPMI medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), and 50 μg/ml gentamicin (Gibco).

Madin-Darby canine kidney (MDCK) epithelial cells (ATCC CCL-34), human embryonic kidney cells (293T; ATCC CRL-11268), human lung epithelial carcinoma cells (A549; ATCC CCL-185), African green monkey kidney epithelial cells (Vero; ATCC CCL-81), and murine fibroblast cells (L929; ATCC CCL-1) were grown at 37°C in air enriched with 5% CO₂ using Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (Gibco) and with 50 μg/ml gentamicin (Gibco). Human HAP-1 haploid wild-type (WT) cells and HAP-1 IFI44 knockout (KO) cells produced using the CRISPR/Cas9 technology were obtained from Horizon Discovery, Inc. These cells were grown at 37°C in air enriched with 5% CO₂ using Iscove’s modified Dulbecco's medium supplemented with 10% fetal bovine serum (Gibco) and with 50 μg/ml gentamicin (Gibco).