Construction And Application Of Interferon Receptor Knockout Cell Line Based On CRISPR/Cas9 System

Objective This study was conducted to construct interferon receptor knockout cell lines using CRISPR/Cas9 technology,aiming to investigate the effect of IFNAR1 knockout on interferon receptor function,to build a technical platform for exploring the mechanism of interferon antiviral in host cells and to improve rotavirus infection titer,and to provide a strong guarantee for subsequent related research.Methods 1.Based on the principle of Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated proteins 9(CRISPR/Cas9)technology,we designed the specific identification of The Lenti CRISPRv2-IFNAR1-sgRNA recombinant plasmid was constructed from the sgRNA(single guide RNA)sequence of the exon region of the interferon alpha and beta receptor subunit 1(IFNAR1)gene,lentivirally packaged and infected with Caco-2 cells,puromycin resistance screening,and monoclonal cell lines were cultured by the limited dilution method.IFNAR1 knockdown was verified by target gene sequencing and Western Blot.2.IFNAR1 knockdown was detected by adding exogenous IFN-β to detect CXC chemokine ligand-10(CXCL10),interferon-stimulated gene 20(ISG stimulated gene 20(ISG20),interferon-stimulated gene 15(ISG15),interferon-induced protein with tetratricopeptide 3(IFIT3),and interferon-stimulated gene 15(ISG15).repeats 3(IFIT3)and m RNA levels of interferon-activating molecules such as myxovirus resistance protein 1(MX1).3.Determination of group H rotavirus(Rotavirus H,RVH)J19 titers in wild-type caco-2 cells by q RT-PCR,and verification of wild-type rotavirus J19 titers by The VP6 protein expression of J19 in wild-type caco-2 cells was verified by indirect immunofluorescence assay,and J19 titer was determined by q RT-PCR after interferon stimulation.4.The viral gene replication after J19 infection in IFNAR1 knockout Caco-2 cell lines and wild type caco-2 cells was compared by q RT-PCR.Results 1.Two IFNAR1 knockout monoclonal cell lines were obtained.The gene sequencing results showed that,among them,a 5 bp deletion occurred in the sixth exon of IFNAR1 in Caco-2-IFNAR1-KO1,and an 18 bp deletion occurred in the seventh exon of Caco-2-IFNAR1-KO2 along with a 1 bp increase;Western Blot results showed that,compared with wild-type Caco-2 cells,Caco-2 IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells showed no expression of IFNAR1 protein compared to wild-type Caco-2 cells.2.Compared to0 ng/ml,the m RNA expression levels of CXCL10 gene,ISG15 gene,ISG20 gene,IFIT3 gene and MX1 gene in knockdown cells stimulated by 50 ng/ml exogenous IFN-β were not significantly m RNA expression levels of CXCL10,ISG15,ISG20,IFIT3 and MX1 genes were not significantly elevated in knockdown cells stimulated with 50 ng/ml exogenous IFN-β compared to wild-type Caco-2 cells.3.Group H rotavirus J19 and group A rotavirus G6P1 were able to replicate and express VP6 protein in wild-type Caco-2 cells.and express VP6 protein in wild-type Caco-2 cells.Meanwhile,the rotavirus J19 titer was significantly decreased in wild-type Caco-2 cells after interferon stimulation.4.Viral replication was increased after group H rotavirus infection with IFNAR1 knockdown Caco-2 cell line compared with wild-type Caco-2 cells;however,there was no significant difference under the present experimental conditions.Conclusions Caco-2 cells can be well used as host cells for rotavirus replication and intracellular interferon antiviral studies,and an IFNAR1 knockout Caco-2 cell line was successfully obtained using CRISPR/Cas9 technology.The downstream molecules dependent on type I interferon receptor activation were significantly inhibited in this cell line,and the preliminary exploration of rotavirus replication after infection of Caco-2 cells provided a platform for subsequent in-depth study of rotavirus infection mechanism,interferon pathway antiviral mechanism and improvement of virus culture efficiency to explore the key factors governing rotavirus culture.