Cell Model Of TNFAIP3/A20 Gene Mutation Was Constructed Using CRISPR/Cas9

Objective:To establish a method for constructing genetic disease cell model using different CRISPR/Cas9 techniques with TNFAIP3/A20 gene mutation as the target of gene editing.To construct cell models expressing TNFAIP3:c.G1428A missense mutations using CRISPR/Cas9 editing techniques.Using the human B lymphocyte line(Lymphoblastoid B cell line,LCL)derived from mutation carriers as the matrix type,the function of the immunodeficiency disease gene TNFAIP3/A20 mutation was preliminarily explored.Method:Two strategies were used to construct cell models expressing TNFAIP3:c.1428G>A mutation.The target mutation was installed into the genomic DNA of the cell,and the mutant gene was highly expressed after the target gene TNFAIP3 is knocked out.(1)PE(prime editing)was used to install the TNFAIP3:c.1428G>A mutation into the genome of HEK293T cells.The efficiency of different PE editing systems was evaluated by the positive mutation RNF2:c.82G>A.Designed and synthesized sgRNA spacer sequence,pegRNA spacer sequence and 3'extension sequence for the target site,and constructed gRNA expression plasmid accordingly.The PE3-GFP editing system was transformed by replacing the GFP element with the Puro element.The new editing system was named PE3-Puro.The sgRNA,pegRNA and PE3-Puro expression plasmids were co-transfected into HEK293T cells to edit the target site.Editing efficiency was determined by PCR-TOPO cloning sequencing and whole exon sequencing.The mutant monoclonal cell lines were selected by the limiting dilution method.(2)The CRISPR/Cas9-based editing which named CBE editor or NHEJ repair pathway was used to knock out TNFAIP3 gene in Hela cells,and then constructed mutant-type and wild-type protein expression vectors for expression regulation.According to the CBE edit box,searched for a target that can intall a stop codon on the TNFAIP3 gene sequence,designed the sgRNA and constructed an expression vector,co-transfect it with the BE4 plasmid into Hela cells,and then select positive cells by puromycin.The two sets of candidate sgRNA sequences were designed and synthesized against the TNFAIP3 gene sequence for knockout.The pX330-mCherry-TNFAIP3 knockout plasmid was constructed and transfected into Hela cells,and positive cells were sorted by flow cytometry.The monoclonal cell lines with gene knockout were selected by limiting dilution method,DNA mutation was detected by PCR-Topo cloning and sequencing,and protein knockout was detected by Western blot.The coding sequence of TNFAIP3 gene was obtained from carrier-derived LCL cells by RT-PCR,and the protein expression vector was constructed by coding sequence.(3)LCL cells derived from immunodeficiency patients and the two cell models were subjected to LPS stimulation.RT-qPCR was used to detect the RNA expression levels of TNFAIP3,TNF-?,RELAnd IL-6 genes,and WB was used to detect the protein expression levels of TNFAIP3/A20.Compared the difference between wild type and mutant type.Result:(1)The TNFAIP3,TNF-?,and IL-6 RNA levels of patient-derived LCL cells without LPS stimulation and TNFAIP3/A20 protein levels after LPS stimulation were lower than normal LCL.(2)The editing efficiency of RNF2:c.82G>A was 30%by the PE3 editor,and it was increased to 100%when using PE3-Puro..The editing efficiency of PE3-Puro installed TNFAIP3:c.1428G>A in HEK293T is 18.58%.The HEK293T cell line with heterozygous mutation of TNFAIP3:c.1428G>A was obtained.Compared with normal HEK293T,there was no difference in the expression of TNFAIP3,TNF-?,RELA and IL-6.(3)NHEJ recombination obtained three Hela cell lines that successfully knocked out the TNFAIP3 gene.Both DNA and protein levels showed that the translation of TNFAIP3 was terminated.There was no significant difference in the effects of mutant and wild-type protein expression regulation on the expression levels of TNF-?,RELA and IL-6.Conclusion:(1)A HEK293T cell model with a heterozygous mutation of TNFAIP3:c:1428G>A and a Hela cell model with overexpression of the mutant gene after TNFAIP3 gene knockout were constructed,which provided a cell platform support for the functional verification of the mutation.(2)The results of mutational function analysis in the LCL cells derived from the patient and the constructed cell model were different,suggesting that the association between different cells and immunodeficiency disease needs to be further explored.