2.6. FACS sorting, single‐cell cloning, and gene editing validation

The top approximately 3% of GFP positive cells were sorted using FACS to enrich for cells that received high levels of the CRISPR/Cas9 constructs. The sorted cells were individually plated into 96‐well plates containing 100 μl per well of cell culture media using FACS sorter. The clones were incubated at 37°C for 3 weeks. The resulting monoclonal colonies were passaged and split to proceed with validation steps.

PCR was used to validate the intended genomic deletion of the ISG15 CDS region. One set of primers lying internally of the sequence to be deleted (nondeletion band: if a band is generated from this pair of primers then the deletion did not occur) and another set of primers upstream and downstream of the sgRNA cleavage sites (deletion band: if a band is generated from this pair of primers then the deletion did occur) were designed (Figure 6 and Table 1). In the absence of deletion, the deletion band is often too large to efficiently amplify. Primers at least 100 bp separated from the predicted cleavage site were used to ensure detection would not be impacted by a small indel at the sgRNA target site. The genomic DNA was extracted from each clone using Invitrogen PureLink Genomic DNA Mini Kit and DNA concentration was measured. Each clone was screened for both nondeletion band and deletion band detection using the following PCR protocol: for each detection, a 25 µl PCR reaction containing 12.5 µl master mix, 0.5 μl forward primer (10 μM), 0.5 μl reverse primer (10 μM), 100 ng gDNA, and H₂O up to 25 μl was run in the thermocycler (98°C for 30 s, 35 cycles of (98°C for 10 s, 60°C for 30 s, 72°C for 1 min), and 72°C for 2 min). The PCR products were then run on 2% agarose gel at 10 V/cm using 1x tris‐acetate‐EDTA (TAE) buffer. The samples were examined for the detection of nondeletion and deletion bands using a Chemidoc (Biorad) (Figure S1) and clones with biallelic deletions were passaged and split for cell banking and further validation analysis. This validation was repeated at a week's interval for quality control. Six independent clones with biallelic deletions were used for the remaining validation steps.

Primers designed for genomic deletion of ISG15 validation

PCR primers design. For the detection of nondeletion and deletion bands

Following the validation of the intended genomic deletion of the ISG15 CDS region, validation at the protein level was done via western blot to ensure that the ISG15 protein is indeed deleted (Figure S2). A total of 20 μl of each cell lysate sample were mixed with SDS loading buffer, separated on SDS‐PAGE gels (BioRad Criterion TGX Precast gels), and transferred to polyvinylidene difluoride membranes. Immunoblotting was performed using the relevant antibodies (anti‐ISG15; Invitrogen). Horseradish peroxidase coupled secondary antibodies were detected with the BioRad Clarity Western ECL substrate. The resulting signals were imaged with a Chemidoc (BioRad) and analyzed by ImageJ.

Following the confirmation of ISG15 deletion, the next validation step consisted of verifying the phenotypic impact of such deletion on virus reproduction. Therefore, triplicates of wild‐type Vero cells and ISG15^−/− Vero cells were cultured and infected at MOI 10 with, on one hand, IAV PR8 and on the other hand rVSV‐GFP. The supernatant for each sample was harvested 24 h postinfection and virus production was quantified via ddPCR (viral genome to quantify the total number of particles) and TCID50 (to quantify the number of infectious viral particles) as previously described (Dzimianski et al., ²⁰¹⁹). We ensure that the cell concentration would not influence the comparison between control and ISG15^−/− Vero cells by using the same cell concentrations for both cases and for all triplicates.