Study On Genome Editing Of Endogenous TRAC Using CRISPR-Cas9 System

Objective: To design three CRISPR targeting sites to knock out the TRAC gene,and identify the better one and investigate the off-target rate of it.To Anaylze the gene editing efficiency in different human cell lines to explore the difference in NHEJ of them.Furthermore,to construct the adenovirus vector carrying CRISPR system to laid the foundation for the research of TCR gene editing in human T cell.Method: 1.Construction,verification and off-target detection of CRISPR-Cas9-TRAC editing plasmids Using the CRISPR website(http://crispr.mit.edu/),three editing sites were designed for TCR alpha C,and the CRISPR-Cas9-TCR editing plasmids were subsequently constructed and transfected into 293 T cells by calcium phosphate reagent.With flow cytometry detection,PCR amplification,and T-A cloning and sequencing,the better g RNA was identified and the editing efficiency was calculated.With websites(http://crispr.mit.edu/;https://chopchop.rc.fas.harvard.edu/index.php;http://cas9.cbi.pku.edu.cn/;http://gt-scan.braembl.org.au/gt-scan/submit),the potential off-target sites for the core sequence of g RNAa were precdicted.10 potential off-traget sites with higher-score were selected for PCR amplification and sequencing analysis with corresponding primers to verify whether there is the off-target editing.2.Comparison of the gene editing in different cell lines of human To further analyze the difference of gene repaire system in different cell lines,the editing plasmid with higher editing efficiency was transfected into different types of human cells(tumor cells and non-tumor cells),and the editing details were detected.The expression level of genes involved in DNA double strand break repair(Ku70,Ku80,DNA-PKcs,Ligase4,XRCC4,XLF,APTX,PNKP,APLF,d P1,Td P2,Artemis,Metnase,WRN,Mre11,pol,pol,TDT)were detected and the expression difference of these genes in different types of cells was analyzed.3.Construction and optimization of adenovirus vector of CRISPR-Cas9 system To further analyze the gene editing effects on T cells by CRISPR-Cas9-TCR system,based on the previous work,we need to construct the adenovirus vector of CRISPR-Cas9-TCR system,and then transfect the CRISPR-Cas9-TCR system into T cells by it.In order to minimize the size of the insert,we choose the p X300 plasmid,which is similar with the p X458 but without the gene of GFP(green fluorescent protein),to the next experiments.We separate the p X330 into two parts for the construction of adenovirus shuttle vectors to increase the success rate.The U6+g RNA fragment was obtained by PCR amplification,and the restriction enzyme sites was added during the amplification.And then constructed the transition plasmid with the fragement and T vevtors,and obtained the p DC315-U6-g RNA adenovirus shuttle vector through a series of reactions between p DC315 and the transition plasmid.In the construction process of p DC315-Cas9 adenovirus shuttle vector,we modified the p DC315 plasmid by adding the Age I restriction enzyme sites to combine with Cas9 sites.Consequently,it was finally obtained by disgesting and reconnection of p DC315 and p X330.Results: 1.The CRISPR-Cas9-TRAC plasmid was successfully constructed and completed the verification and detection of the off-target rate.Based on the p X458 plasmid,we constructe the gene editing plasmids(p X458-site1?p X458-site2?p X458-site3)for the target sites of TCR alpha C,and transfected them into the 293 T cells by calcium phosphate reagent.The flow cytometry detection results revealed that the transfection efficiency rates of p X458-site1,p X458-site2 and p X458-site3 were 38.4%,30.5% and 37.8%,respectively.After the trafection of these three type cells for 72 h,extracted the genomic DNA of them.Through the PCR amplification and the sequencing of PCR products,we know that the p X458-site2 plasmid was editing successfully,and then followed by T-A cloning and sequencing,35 monoclonal tested,8 occurred gene editing,when considered the transfection rate,the efficiency is about 74.7%.The PCR amplificated and sequencing results suggested that there were no gene edition among the 10 top off-target sites,which indicating that the p X458-site2 possess significant targeting.2.There are gene editing differentiation among the cell lines by CRISPR-Cas9 system The flow cytometry detection results revealed that the transfection efficiency rates of HEK-293 and SW480 were 29.4% and 21.9%,respectively.After the trafection of these cells for 72 h,extracted the genomic DNA of them.Through the PCR amplification and the sequencing of PCR products,and then followed by T-A cloning and sequencing,in the 293 cell lines,25 monoclonal tested,4 insert occurred and all a bases,the insertion location are three bases uptream PAM,the efficiency is about 68%.In the SW480 cell lines,56 monoclonal tested,4 occurerd gene edition,2 insert occurred and all a bases,the insertion location are three bases uptream PAM,the efficiency is about 32.7%.Download the genes involved in the NHEJ process(Ku70?Ku80?DNA-PKcs?Ligase4?XRCC4?XLF?APTX?PNKP?APLF?Td P1?Td P2?Artemis?Metnase?WRN?Mre11?poll?polm?TDT)from the Pubmed website,designed the corresponding upstream and downstream primers,analyzed the expression difference of these genes,the results revealed that there are difference in the expression these genes in different types of cells,laying the foundation for the further ralative study.3.The two adenovirus vector of CRISPR-Cas9-TCR system was successfully constructed.The U6+g RNA fragment was obtained by PCR amplification,obtained the p DC315-U6-g RNA adenovirus vector by disgesting and reconnection.Since the obsence of Age I restriction enzyme sites,only the p X330 digested by the Age I to get the Cas9 fragment through the screening of two methods for construction for the plasmids,the p DC315-Cas9 adenovirus vector can be obtained succecssfully.Conclusion: The TCR alpha C gene editing plasmids have been constructed successfully,and proved that the p X458-site2 plasmid with higher editing efficiency and lower off-target rates.The p X458-site2 have been transfected into different lines of human by lipo3000 successfully,through the gene editing results it can found that there are large propotion with A/T insert in the same site,and there are some genes which involved in the NHEJ process with differential expression between the tumor cell lines and non tumor cell lines of human.And we constructed the adenovirus vector of CRISPR-Cas9-TCR system(p DC315-U6+g RNA,p DC315-Cas9)successfully,laying the foundation for the subsequent modification of TCR gene in T cells.