Research On Targeting Tumor Mutant Labeled Cells And Inducing Immune Recognition Based On CRISPR

Objective:CRISPR/Cas9 technology was used to identify and edit specific gene mutations in cancer cell lines,insert antigen epitope peptide sequence into specific mutation sites of cancer cells,and make exogenous epitope peptide expressed in cancer cells and presented on the surface of cell membranes to be recognized by various immune cells,causing a wide range of immune responses.Study the edit treatment to CRISPR/Cas technology based on target gene,can cause specific killing immune cells to exogenous antigen peptide.It was also discussed whether CRISPR/Cas9 technology could accurately identify normal cells and mutant cells at the target site,so that antigen peptides could be accurately located in mutant cells.For the follow-up for specific recognition of cancer cell killing experiment basis mutation.Method:TP53,a common mutation gene in cancer cells,was selected to target the CDS region mutation of TP53 gene.Mutations in HEK-293,HEK293-T,K562,Jurkat,HeLa and SW480 cell lines were screened by RT-PCR sequencing products.The core sequence of sgRNA was designed to construct pX458 targeted editing plasmid by using the target editing site screening tool provided by CRISPR website(http://crispr.mit.edu/).Three mutant cells were targeted by electroporation,and the suitable electroporation method for suspension cells was explored,and the editing rate of the targeted editing plasmid was obtained.In addition,we also transfected B cells without single base mutation of A cells with edited plasmids targeting A cells by:(1)introducing a 1 nucleotide mismatch artificially to the designed cutting site of sgRNA sequence;(2)transfecting B cells without single base mutation of A cells with edited plasmids targeting A cells.To verify the effectiveness of this targeted strategy for identifying single base mutations.Finally,we construct different homologous recombination templates.Through different forms of homologous recombination template,the pX458 edited plasmid and template were co-transformed by electrophoresis to the selected K562(myeloblastoma cell line).To investigate whether HBVs1 antigen peptide sequence can be inserted into target site and expressed.Result:In the first part,we confirm that HEK-293,Hela and SW480 in our laboratory are wild-type cells of TP53 gene by comparing with reference sequences,but there are different mutations in CDS region of genes in HEK-293 T,Jurkat and K562 cell lines.Allele mutation(A T base substitution)was found at 840 site of CDS region in 293 T cells,which changed the 280 th amino acid(S R).In Jurkat cells,"G" at position 1083 had base deletion and 200 bp deletion at position 177.A single base "C" insertion occurred between position 406 C and position 407 A in K562 cells.After considering the mutation,sgRNA was designed.Finally,pX458-K562 sgRNA,pX458-293 T 1sgRNA,pX458-293 T 2sgRNA were successfully constructed.In the second part,through the optimization of plasmid and electroporation conditions,the transfection efficiency of pX458 plasmid D1(K562 215 v 3ms;Jurkat 150 V 3ms)suitable for K562 and Jurkat cells in our laboratory can reach: K562 48.4%.In addition,HEK-293 T was transfected by calcium phosphate transfection,the efficiency was 34.7% and 21.3%.In addition,we validated the effectiveness of the targeted strategy for identifying single base mutations in this part of the experiment.In the third part of the experiment,we selected the surface protein sequence of hepatitis b virus and the "WLSLLVPFV" peptide sequence,named HBVs1,as the epitope sequence of TP53 mutation site inserted into K562 cells through online analysis.we successfully verified that the ssDNA template inserted K562 mutation target in co-transformation.Sequencing peaks show the sequence of HBVs1 immediately after the cleavage site in the left arm.The expression of K562 with red fluorescence was also observed after co-transformation of the denoter plasmid with red fluorescence labeling.In order to present the inserted epitope,we successfully constructed a pair of lentiviral shuttle plasmids expressing HLAA020101 and B2 m molecules,which laid the foundation for subsequent killing experiments.Conclusion:Using CRISPR/Cas9 technology,it is feasible to identify specific gene mutations in cancer cell lines,target edit and insert antigen peptides into target sites.However,the efficiency of insertion integration still needs to be further improved.