Construction Of Recombinant CHO Cell Line With Gene-directed Editing Based On CRISPR/Cas9 Technology

Background Chinese hamster ovary(CHO)cells are the preferred host for the production of recombinant therapeutic protein.Establishing stable Chinese Hamster Ovary(CHO)cells producing monoclonal antibodies(m Abs)usually pass through the random integration of vectors to the cell genome,which is sensitive to gene silencing.This method usually accompanied by the phenomenon of transgene silencing and transgene expression attenuation caused by "position effect",for it can not control the insertion site of the genes of interest(GOI).Cytidine monophosphate-N-acetylneuraminic acid hydroxylase(CMAH)in CHO cells can produce N-glycolylneuraminic acids(Neu5Gc)which elicits the immune response in the human body.Therefore,it is vital to silence the expression of CMAH in CHO cells.Genome editing using Clusterd regularly interspaced short palindromic repeats(CRISPR)/Cas9 system is becoming a robust tool for targeted and stable mutagenesis of DNA.Objective To knock out CMAH using CRISPR/Cas9 technology to construct CMAH deficient CHO cell expression system.Methods 1.Bioinformatics analysis: The CMAH gene sequence of CHO cells were searched on NCBI.The eighth coding sequence of CMAH was determined as the target sequence.sg RNAs for CMAH were designed using the online software ZIFIT.2.Vector construction:(1)sg RNAs were designed according to the target site CMAH,and constructed the CRISPR/Cas9-sg RNA system.(2)The GOIs(EGFP gene,EPO gene, EPO-FC gene)was inserted into the expression cassette of the MAR expression vector in the laboratory,followed by designing the homology arm sequence and constructing the homology arm on both sides of the GOIs to construct donor vector.3.Cell transfection and screening: CHO cells were co-transfected with the donor vector and CRISPR/Cas9-sg RNA system by the liposome,and the cells transfected with the donor vector was used as the control;The stable cell lines were obtained through screening under pressure of Blasticidin S and puromycin.Monoclone cells were obtained by inoculating 1 cell/well in 96 well plate with limiting dilution.4.Verification of cells with site-specific integration: Genomic DNAs of CHO cells were extracted as PCR template and PCR was performed with specific primers of CMAH gene.The PCR products were sequenced to confirm site-specific integration of GOIs occurred.5.m RNA expression detection: Total RNA was extracted from mutant cell lines and c DNA was systhesised by using an reserve transcription system.Then,the m RNA expression levels of CMAH gene and GOIs were detected by real-time quantitative PCR.6.Recombinant protein expression: Flow cytometry was used to analyze the EGFP expression of cell line,and the relative protein quality(glycotype)was detected by UPLC.Results 1.The CRISPR?Cas9 system and donor vector were successfully constructed.2.The recombinant cell pools were obtained and the recombinant monoclonal cells were achieved by limiting dilution.3.We targeted exogenous genes(EGFP gene,EPO gene,EPO-Fc gene)into CMAH loci successfully through site specific integration mediated by CRISPR/Cas9 and the targeting ratio was from 12.5% to 19.2%.4.We found that the expression level of EGFP transgene in the site integrated cell line was not higher than that of random integrated cell line.However,the production of EGFP in the CMAH site integrated cell line was more stable and more uniform.5.Compared with the random integration,the glycosides proportion of G0F-GN,G0,Man-5,G2FS1,G2FS2,FA4G4LAC4S4 in EPO was increased,while the glycosides proportion of G0 F,FA3G3S3,F(6)M5A1G(4)1Ga(3)1 was decreased.Conclusion 1.Compared with wild type CHO-S and targeted integration cells,we found that CMAH site is not essential to cell growth and could be engineered without substantial interference to cell characteristics.2.Compared with randomly integrated cells,inserting the GOIs at the CMAH site not increased protein expression,but the protein expression is more stable and more uniform;Compared with EPO expressed in random integration cells,the glycotypes of EPO expressed in CMAH site were changed.