Celltiter glo luciferase assay

100 TCID50 of SARS-CoV-2 (isolate USA-WA1/2020) was incubated with 2-fold serial rabbit serum dilutions in a round bottom plate at 37°C for 1 hour. The virus-antibody mixture was then added to a 96-well plate with 5x10⁴ Vero E6 cells. After 1 hour the mixture was removed and replenished with fresh MEM containing 2% FBS. Cells were incubated at 37°C for an additional 72 hours, then cytopathic effect (CPE) was measured by CellTiter-Glo luciferase assay (Promega). Luciferase abundance was determined using Veritas luminometer. The end-point titers were calculated as the last serum dilution resulting in at least 50% SARS-CoV-2 neutralization.

PP-spheroids (HUES4) at passage 3 were dissociated as described above. A Bravo liquid handling robot (Agilent) was used to deposit 8 µl of cell suspension in all but 3 wells of a 96-well OptiPlate (PerkinElmer). GFR-Matrigel mixture without cells was added to the last three wells, to serve as a negative control. For the first 48 h, PP-spheroids were cultured as normal, in an expansion medium. After 48 h expansion medium was removed, wells were washed with 1× PBS twice, and different media were added in triplicates, including 26 conditions and 3 controls (details on Supplementary Table 3). The medium was again changed at day 5 and 8. After a total of 10 days in culture, Cell Titer-Glo luciferase assay (Promega) was performed according to the manufacturer’s instructions, and luminosity was measured in the LUMIstar omega (BMG Labtech). The validation experiments were performed in Nunc 4 well plates with both the HUES4 and SBAD3.4 cell lines, with the same timeline used in the original screening experiment, except for Infigratinib, which was only added from day 9–10. Cells were collected at days 5 and 10 of culture for EdU quantification. Validation in fetal spheres was performed in 8-well µ-slides (Ibidi).

Cryopreserved HeLa cells were thawed and reconstituted in DMEM with 10 % FBS at a density of 5 × 10⁵ cells per mL, then plated in 24 well plate (1 mL per well) 4 h prior to transfection. Transfection was performed using Lipofectamine 2000 (Invitrogen, United States) under the following conditions: 5 μL Lipofectamine 2000 (1 mg/mL), 300 ng of Cpf1 expression vector, 500 ng of the crRNA plasmid, 500 ng of corresponding pGFP-SSA target construct, were mixed in OptiMEM I (Gibco, United States) to a final volume of 500 μL. The pGFP-SSA 5′-TNNa-3′ PAM plasmid library experiments were performed with a lower quantity of nuclease expression vector (100 ng). After 2 days incubation, image of fluorescent cells, expressing GFP protein, were obtained using a Nikon Eclipse confocal microscope (Nikon, Japan). The cell surviving was measured using Cell-titer-Glo luciferase assay (Promega, United States). The fluorescence was characterized using a Filter max F5 reader Multimode (Molecular Device, United States). For each transfection experiment a pGFP construct was co-transfected under the same conditions as a GFP signal stability control, and a pGFP-SSA EcoRI linearized as a reference for the percentage of fluorescence calculation. Duplicates or triplicates transfection were performed during the same experiment for a better quantitative reproducibility.