## 3 Results
### RABV Production Was Higher in MRC-5f\({}^{\text{IFNAR1}-}\) Cell Line  
To test the effects of genetic IFN signaling pathway defectiveness in MRC-5 cells affecting RABV yield, GenCRISPR(tm) gene editing technology was used to establish an IFNAR1-silent MRC-5 stable cell line (MRC-5f\({}^{\text{IFNAR1}-}\)) and a non-targeting control MRC-5 stable cell line (MRC-5\({}^{\text{NC}}\)). Based on human IFNAR1 genomic sequences (GeneBank ID 3454), three targeting gRNAs were designed for GenCRISPR(tm) system (Table 4). By the transient transfection of GenScript CRISPR single-guide RNAs Cas9 in MRC-5 cells, the cleavage efficiency of three samples was 44%, 40%, and 48%, which was revealed by sequencing trace analysis using CAT CRISP RAB (Table 4). The mutations were identified by Sanger sequencing (Figure 4A). Three MRC-5f\({}^{\text{IFNAR1}-}\) cell lines, MRC-5\({}^{\text{NC}}\), and MRC-5 cells RNA were extracted, and the expression of IFNAR1 mRNA was detected by qRT-PCR, respectively (Figure 4B). The IFNAR1 mRNA expression in each of the three MRC-5f\({}^{\text{IFNAR1}-}\) cell lines decreased by 62%, 73%, and 57% compared with that in MRC-5 cells. The decrease in mRNA expression level caused by mutation is likely due to NMD (nonsense-mediated mRNA decay) or NSD (no-stop decay) [36; 37; 38]. There was no significant difference between MRC-5\({}^{\text{NC}}\) and MRC-5 cells. The MRC-5f\({}^{\text{IFNAR1}-}\), 2, 3, or MRC-5 cells were infected with RABV, and then endogenous IFN-\(\beta\) and OAS1 mRNA expression was examined by qRT-PCR at 24 hpi (Figure 4C). The IFN-\(\beta\) and OAS1 mRNA expression in each of three MRC-5f\({}^{\text{IFNAR1}-}\) cell lines decreased significantly compared with that in MRC-5 cells. The viral RNA in the culture supernatant of MRC-5f\({}^{\text{IFNAR1}-}\) or MRC-5 infected cells were also tested by qRT-PCR at 96 and 168 hpi. Viral production was significantly improved  
Figure 3: IFNAR1 antibodies enhanced RABV production in MRC-5 cells. MRC-5 cells were infected with the PM RABV strain at an MOI of 0.05. Vero cells were infected with the aG RABV strain at an MOI of 0.001. The IFNAR1 monoclonal antibodies \(\alpha/\beta\)-IFNAR1 and \(\alpha\)-IFNAR1 were incubated with MRC-5 cells 2 h before virus infection at a concentration of 1 ng/mL. (**A**) Total RNA from cells was extracted at 24 hpi, and the expression of IFN-\(\alpha\), IFN-\(\beta\), and OAS1 mRNA was detected by qRT-PCR. Viral RNA (**B**) and titer (**C**) in the culture supernatant were detected by qRT-PCR and FFU assays at 168 hpi. *: RABV infection; * \(p\) < 0.05; ** \(p\) < 0.01; *** \(p\) < 0.001; *** \(p\) < 0.0001; ns \(p\) \(\geq\) 0.05.  
in each of three MRC-5F\({}^{\text{IFNAR1}-}\) cell lines compared with MRC-5 cells (Figure 4D). The MRC-5F\({}^{\text{IFNAR1}-}\)-2 was the most productive cell line with a 6.5-fold increase in yields. These data suggest that the IFNAR1-defective cell lines can be used for RABV-inactivated vaccine production. Use of IFN Inhibitor or MRC-5F\({}^{\text{IFNAR1}-}\) Cell Lines Enhanced RABV Production in Pilot Scale-Up Experiments  
We observed that three efficient approaches enhanced RABV production in MRC-5 cells. To further estimate their application in industrialization, IFN inhibitors and MRC-5F\({}^{\text{IFNAR1}-}\) cells were tested in pilot scale-up experiments for improving RABV production. \begin{table}
\begin{tabular}{c c c} \hline \hline
**gRNAs** & **Sequence** & **Cleavage Efficiency** \\ \hline MRC-5F\({}^{\text{IFNAR1}-}\)-1 & CTGGAGCCACCTGAACTTGAAA & 44\% \\ MRC-5F\({}^{\text{IFNAR1}-}\)-2 & TTCCATGATGCTGTACG & 40\% \\ MRC-5F\({}^{\text{IFNAR1}-}\)-3 & AGTGGATAATCCTGGATAC & 48\% \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Sequences of IFNAR1 guidance RNA (gRNA) and evaluation of gRNA cleavage efficiency. Figure 4: RABV production was enhanced in MRC-5F\({}^{\text{IFNAR1}-}\) cells. (**A**) Genomic DNA from three MRC-5F\({}^{\text{IFNAR1}-}\): gRNAs cell lines was extracted.