One-step Hybrid Multi-gene Knock-in And Knock-out Editing System Development And Study In Porcine Cells

With the rapid development of gene editing technology,CRISPR/Cas9 system has been widely used in targeted breeding,human disease model construction and gene therapy.As an important agricultural animal,pigs are more similar to humans in terms of physiology and anatomy,and are the best choice for preparing human disease models.In recent years,researchers have used CRISPR technology to prepare a variety of disease model pigs,which has strongly promoted the research of human diseases,especially genetic diseases.At present,disease model pigs are relatively mature for target screening and new drug development of single gene genetic disease treatment;however,the preparation of multigene genetic disease model pigs is greatly limited by the lack of efficient multi-gene manipulation technology,especially multi-gene knock-in and knock-out systems.To address the lack of multi-gene manipulation technology,this study successfully developed a CRISPR system-mediated one-step hybrid multi-gene knock-in and knock-out editing system,and used this technology to knock in PAX3-FOXO1 fusion protein sequence at PAX3 locus and zs Green green fluorescent protein sequence at PAX7 locus in 3D4/21 cells by separate dosing screening method with Cas9 expression sequence,and knocked out TP53 gene and FOXO1 gene simultaneously,thus successfully constructing rhabdomyosarcoma cell model and providing a new approach for the creation of multi-gene genetic disease model pigs.The results of the study are as follows:1.SgRNAs targeting PAX3,PAX7,TP53,and FOXO1 genes were designed using sgRNA design software.SgRNAs were packaged and infected with PK15-Cas9 cell lines by lentivirus,and cells were collected 72 h after infection.genomic DNA was extracted and sequences near the sgRNA targets were amplified,and sequences were assessed using sanger sequencing and T7 EN 1 assay.SgRNA activity was assessed using sanger sequencing and T7 EN 1 assay.One sgRNA with the highest efficiency was finally selected for each gene and could be used for subsequent studies.2.After determining the sequence of sgRNA,PAX3-KI-donor and PAX7-KI-donor vectors were designed and constructed.The main components in PAX3-KI-donor were PAX3-FOXO1 fusion protein sequence,puromycin resistance gene sequence(puro R),U6-sgRNA-csy4 sequence(Csy4 tandem targeting four gene sgRNA sequences).the main components in PAX7-KI-donor are zs Green green fluorescent protein sequence,Cas9 protein sequence,and insecticide-resistant gene sequence(blasticidin).In cells,Cas9 transcriptional translation can bind to sgRNA to target two endogenous sites,PAX3 and PAX7,and then cells use homologous recombination to precisely integrate the exogenous knock-in fragment at the target site.3.In order to assess the tolerance properties of cells to antibiotics,we mapped the way of adding antibiotics.After co-transfecting PAX3-KI-donor vector and PAX7-KIdonor vector into PFF cells and 3D4/21 cells,by comparing the way of adding two antibiotics simultaneously or separately in the cell culture medium,we found that when two antibiotics were added simultaneously,PFF cells and 3D4/21 cells all died within a short period of time,indicating that adding two antibiotics simultaneously.The cells were found to die within a short period of time when both antibiotics were added simultaneously,indicating that the cell status was affected more when both antibiotics were added simultaneously,while the cells could grow normally when both antibiotics were added separately in the cell culture medium.Therefore,in the subsequent screening process,we used the form of adding antibiotics to the cell culture medium separately to screen the cells.4.To test whether the system can obtain cells with both double knock-in and double knock-out genes.We transfected PAX3-KI-donor vector and PAX7-KI-donor vector into3D4/21 cells,screened the cells using a strategy of adding antibiotics separately,and obtained monoclonal cells and genotyped them.The results showed that among the 58 monoclonal cells selected,#12,#13,#32,#50,#51 monoclonal cells PAX3 locus knock-in was successful with a knock-in efficiency of 8.6%;#13,#32,#50,#51 and 18 monoclonal cells PAX7 locus knock-in was successful with a knock-in efficiency of 31.0%;all monoclonal TP53,FOXO1 genes were knocked out.Double knock-in and double knockout(quadruple gene editing)monoclonal successes were #13,#32,#50,#51,with an efficiency of 6.8%.5.To test the safety of the knock-in system developed in this study,the selected sgRNAs need to be evaluated for off-target.For the four sgRNAs used in this study,offtarget sites were predicted by the CRISPR-offinder website,and a positive clone was randomly selected,and the potential off-target sites predicted by the website were amplified,and sanger sequencing was used to detect whether editing occurred at these sites.The results showed that no editing occurred for any of the predicted potential off-target sites.These results demonstrate that the one-step hybrid multiple knock-in and knock-out editing system developed in this study is safe.In summary,this study established a one-step hybrid multi-gene knock-in and knockout editing system based on CRISPR/Cas9 technology,and applied the system to successfully establish a rhabdomyosarcoma disease cell model in a porcine cell line.The results of this study provide a new idea for the preparation of polygenic disease model pigs and a new gene editing tool for the application of gene editing breeding technology in pig genetic improvement.