Construction Of Glycosylases CMAH And GGTA1 Genemutants In CHO Cells Using CRISPR/Cas9

Background and Objective The production of therapeutic recombinant proteins is an important part of biopharmaceuticals,and recombinant glycoproteins are used to treat many diseases.Chinese hamster ovary(CHO)cells are the most commonly used mammalian cell line,which are used as hostto producetherapeutic recombinant glycoproteins that are close to the human-like glycosylation of glycoproteins.The biggest difference between glycosylation of human and CHO cells is that CHO cells have twoglycosylases,namely ?-1,3-galactosyltransferase(GGTA1)and CMP-N-acetylneuraminic acid hydroxylase(CMAH),which have been deleted in humans.When using CHO cells to produce the therapeutic glycoproteins,?-1,3-galactose(?-gal)produced by GGTA1 and N-glycolylneuraminic acid(Neu5Gc)produced by CMAH can lead to immune response in the human body.In this study,CRISPR/Cas9 gene editing technology was used to genetically engineer CHO cells,and CHO cell lines with mutated CMAH and GGTA1 genes were constructed to improve therapeutic safety of therapeutic recombinant glycoproteins.Methods 1.Bioinformatics analysis: The CMAH and GGTA1 gene sequences of Chinese hamsters were searched on NCBI.CDS8 of CMAH and CDS9 of GGTA1 were determined as target sequences.CRISPR/Cas Nucleases of online software ZIFIT were selected.The determined target sequence was copied and pasted onto a web page to design the sg RNA of CMAH and GGTA1.Design Reporter sequencesbased on sg RNA sequence position? 2.Construction vectors: The synthesized sg RNAs of CMAHand GGTA1 were digested and ligated with p GL3-U6 plasmid separately.The synthesized Reporter sequences of CMAH and GGTA1 were digested and ligated with pm Cherry-EGFP plasmid respectively.The plasmids were then transformed into E.coli DH5? competent cells.The plasmid DNAs were extracted and identified by agarose gel electrophoresis after enzymes digestion.3.Cell transfection: p GL3-U6-CMAH-sg RNA,pm Cherry-EGFP-CMAH-Reporter,Cas9 and p GL3-U6-GGTA1-sg RNA ? pm Cherry-EGFP-GGTA1-Reporter ? Cas9 wererespectively co-transfected into HEK293 T cells using lip3000 in order to verify the efficiency of sg RNA cleavage.p GL3-U6-CMAH-sg RNA with highly efficient was selected to transfect target cell CHO-S.p GL3-U6-GGTA1-sg RNA with high cutting efficiency was selected to transfect the CHO-S cells wtth CMAH gene mutation.4.Single cell cloning screening: Six CHO-S monoclonal cells were sorted by flow cytometry sorter.CHO-S monoclonal cells with CMAH gene knockout strains were selected to further mutate GGTA1 gene.Six CHO-S monoclonal cells were obtained by flow sorting and expanded.5.Gene sequencing of mutant cell lines analysis: PCR primers were designed at both ends of the target sequence of CMAH and GGTA1.Genomic DNAs from CHO-S monoclonal cellswere extractedand amplified by PCR,then subjected to Sanger sequencing.6.Detection of m RNA expression in mutant cell lines: RNA of target cells was extracted and reverse transcribed into c DNA.Q-PCR primers for CMAH and GGTA1 genes were designed to detect the m RNA expression levels of the genes.7.Target gene expression:The vectorp IRES-neo-EPO containing Erythropoietin(EPO)was transfected into the two CHO mutant cells.The expression of EPO was detectedby western blot.Results 1.The CMAH gene cleavage vector p GL3-U6-CMAH-sg RNA and the reporter vector pm Cherry-EGFP-CMAH-Reporter were successfully constructed using the CRISPR/Cas9 technology.2.The GGTA1 gene cleavage vector p GL3-U6-GGTA1-sg RNA and the reporter vector pm Cherry-EGFP-GGTA1-Reporter were successfully constructed using the CRISPR/Cas9 technology.3.The monoclonal CHO-S cells with CMAH and GGTA1 gene mutation were obtained by flow sorting technique.4.Mutantcell 1-5 have one base C deletion at position 99546 bp of the CMAH gene and one base T between 68386 and 68387 bp of the GGTA1 gene.5.Compared with wild-type CHO-S cells,the expression levels of CMAH and GGTA1 m RNA in the mutant cells 1-5 were significantly reduced.6.In contast with wild-type CHO-S cells,mutantcells1-5 have a strongerproliferative capacity and are capable of expressing EPO proteins.Conclusions 1.The CHO-S cell subline 1-5 with CMAH and GGTA1 gene mutation were successfully constructed by CRISPR/Cas9 technology.2.The mutantcells1-5 are heterogeneous with wild-type CHO-S in proliferation.3.The mutantcells1-5 can express the glycoprotein of interest.