Studies On Site-specific Integration And Stable Expression Of The Exogenous Protein Within A Novel Site On CHO Cell Genome

Chinese hamster ovary(CHO)cells are widely used production cells in the biopharmaceutical field.The traditional method of constructing the recombinant cell lines is to integrate the target gene into the chromosome genome randomly,and then go through many times of pressure screening.The construction process is time-consuming and laborious,and often due to the instability of integration sites,with the increase of passage times,the number of unproductive cell clones gradually increases,resulting in the decline of product yield.It is expected to shorten the construction cycle and solve the problem of stability by the finding of the stable integration sites and integration of the foreign genes into the stable sites of CHO cell genome by site-specific integration.In the early stage of the project,six potential site-specific expression sites were found by lentivirus tracing and chromosome walking technology.In this study,it was achieved that the site-specific integration and expression stability of exogenous genes within the range of 148052?148157 bp on chromosome NW₀03614241.1 site using CRISPR/Cas9 gene editing technology,laying a foundation for the further development and utilization of this site.The recombinant Bak/Bax double knockout CHO-K1 cell line 1g11,which was constructed by lentivirus transfection technology,was used as the research object,the expression of Zsgreen1 gene was characterized by an inverted fluorescence microscope and a flow cytometer to quantitatively analyze the average fluorescence intensity of the cells.It was found that all of the lgll cells had stable green fluorescence intensity after 60 consecutive passages both in adherent culture conditions and suspension culture conditions,which proved that this site of CHO cells can stably express the Zsgreen1 gene.To find potential target positions within the range of 148052?148157 bases in NW₀03614241.1 site,the CCTOP CRISPR/Cas9 online prediction system was used.In this study,a target sequence 5'-ACCCTTGTGCCCCAAAGACAGGG-3' was selected to construct an sgRNA plasmid,then transfect the Bak/B ax double knockout CHO-K1 cell line(Ie3).Result of agarose gel electrophoresis and sequencing verified that sgRNA can be effectively edited at the target site,and the editing efficiency was about 69.5%,indicating that the NW₀03614241.1 site can be edited.The elements designed on the EGFP donor plasmid including 5'homology arm,enhanced green fluorescent protein(EGFP)gene expression cassette,puromycin(Puro)gene expression cassette and 3'homology arm.Using CRISPR/Cas9 technology,three-plasmid co-transfection integrated the EGFP gene and Puro gene between the 5'homology arm and the 3'homology arm into the Ie3 genome by homology repair.After puromycin pressurization screening,flow cytometry sorting,and PCR amplification verification,3 recombinant monoclonal cell lines EGFP-Ie3-19,EGFP-Ie3-48,EGFP-Ie3-51 were obtained,which successfully knocked into the whole EGFP gene,and the positive monoclonals were all heterozygous.It was verified by sequencing that the knock-in positions were all at base 118097 of NW₀03614241.1.Suspension acclimatization of positive monoclonal strains,then using flow cytometry observed the expression of EGFP gene.It was found that all of the positive monoclonal cell lines could stably express green fluorescent protein in the process of continuous suspension subculture for 60 generations,indicating that the EGFP gene can be site-specifically integrated and stably expressed at this site.To construct the HSA donor plasmid,the human serum albumin(HSA)gene was added to the EGFP donor plasmid by enzymatic digestion and connection based on restriction enzyme sites Kpn I and Xma I.Similarly,the three plasmids were co-transfected to knock-in the secreted protein HSA gene in the Ie3 genome.After puromycin pressurization screening,flow cytometry sorting,Dot blot screening,PCR amplification and Western blot verification,3 recombinant monoclonal cell lines HSA-Ie3-4,HSA-Ie3-52,HSA-Ie3-61 were obtained,which successfully knocked into the whole HSA gene.And the positive monoclonals were all heterozygous.Result of sequencing verified that the knock-in positions were also at base 118097 of NW₀03614241.1.Suspension acclimatization of positive monoclonal strains,then batch culture and quantitative detection of HSA protein expression level.It was found that the growth and HSA expression of HSA-Ie3-4 was the best,which was 13.6 mg·L-1,so selected HSA-Ie3-4 to be fed-batch culture,finally HSA expression was 14.23 mg·L-1.Afterwards,sodium butyrate and ferric citrate were added to the CD CHO serum-free medium for preliminary optimization.It was found that when the concentration of sodium butyrate was 3 mmol·L-1,the protein expression was 19.35 mg·L-1,which had the best effect and improved 36.0%;when the added concentration of ferric citrate was 0.5 mmol·L-1,the protein expression is 16.35 mg·L-1,the effect is the best,with an increase of 14.9%.The results of the study showed that both EGFP and HSA gene can be site-specifically integrated and stably expressed within the range of 148052?148157 bp on chromosome NW₀03614241.1.This site in CHO cell has the potential to become a new stable expression hotspot.