Screening And Verification Of Stable Expression NW-003613781.1 In CHO-K1 Cell Genome

Chinese hamster ovary（CHO）cell is the most widely used expression system in biopharmaceutical field to express various therapeutic protein and antibodies.Random integration is a traditional method to construct recombinant cell lines.Although this technology is more mature and simple,the integration sites of the constructed cells are uncertain,which easily leads to high-level cloning variation,and the construction process is time-consuming,costly and inefficient.The expression cell lines constructed by the site-specific integration are predictable,which can effectively reduce the level of clonal variation,and shorten the construction period of stable recombinant expression cell lines.However,the construction of stable expression strains by site-specific integration requires a stable site with clear information.In the early stage,the research group obtained several CHO-K1 cell lines with potential stable expression ability through the random integration of Zsgreen1 reporter gene through lentivirus infection.In this study,the above-mentioned cells were used as research materials to obtain information of several stable expression sites,and one of them was selected to construct a stable platform cell line containing Bxb1 recombinase landing pad by CRISPR/Cas9 technique,and the gene recombination mediated by Bxb1 recombinase was used to realize the site-specific integration of foreign genes.（1）CHO-K1 cell lines with potential stable expression site,which was randomly integrated with zsgreen1 reporter gene by lentivirus infection in the early stage of the research group,was cultured for 20 generations and then suspension cultured for 50 generations.The expression of green fluorescent tag protein Zsgreen1 was analyzed by inverted fluorescence microscope and flow cytometry.Four cell lines which could stably express Zsgreen1 protein were obtained:CHO-K1-1d2,CHO-K1-1b7,CHO-K1-2f7 and CHO-K1-2d9 cells.（2）The integration sites of zsgreen1 gene in the above four cells were analyzed and verified by genome walking technique.The genomic information of stable expression sites in the four CHO cells were separately located between the 1159463th base and the 1159467th base of genome NW-003614092.1,between the 1497183th base and the 1497187th base of genome NW-003613781.1,between the 101420th base and the 101424th base of genome NW-003613756.1 and between the 103431th base and the 103435th base of genome NW-003614889.1.（3）The sequence between the 1497083th base and the 1497287th base of the genome NW-003613781.1 was analyzed by the CCTOP CRISPR/Cas9 online prediction system,and the target sequence recognized by CRISPR/Cas9 system is:5’-GCACTATATGCACATGCCAGTG G-3’,then the sgRNA expression plasmid targeting the sequence was constructed which was co-transfected with the Cas9 protein expression plasmid into CHO-K1 cells.After T7E1digestion and sequencing verification,it was found that the sgRNA can guide the Cas9 protein to break the DNA double-strand at the target site,which indicated the sequence between the1497083th base and the 1497287th base of the genome NW-003613781.1 was editable.（4）Sg RNA expression plasmid,Cas9 protein expression plasmid and enhanced green fluorescent protein（EGFP）donor plasmid were co-transfected,and the expression EGFP gene carried the attP-specific site of the Bxb1 integrase system and puromycin resistance gene was site-specifically integrated at the target site by CRISPR/Cas9 technology-mediated homology-directed repairing.After puromycin screening,flow cytometry sorting and PCR verification,5 positive cloned cell lines with site-specific integration were obtained:K1-1b7-EGFP-17 is homozygous and K1-1b7-EGFP-29,K1-1b7-EGFP-35,K1-1b7-EGFP-37 and K1-1b7-EGFP-43 are heterozygous.K1-1b7-EGFP-17 and K1-1b7-EGFP-29 cells were selected and cultured in suspension for 50 generations.The expression of EGFP protein was analyzed by flow cytometry,and it was found that the selected cells could stably express EGFP protein,which indicated that a stable platform cell line containing Bxb1 integrase landing pad（LP）for site-specific recombination was successfully established.（5）IFNβ-HSA fusion protein donor plasmid containing attB-specific site recognized by Bxb1 integrase was constructed,and then the site-specific integration of IFNβ-HSA fusion protein gene and bleomycin resistance gene expression cassette was realized through DNA recombination between attP and attB sites.After bleomycin screening,flow cytometry sorting,Dolt blot,PCR verification and Western blot,6 positive clones were obtained:K1-1b7-IFNβ-HSA-2.K1-1b7-IFNβ-HSA-11,K1-1b7-IFNβ-HSA-20,K1-1b7-IFNβ-HSA-26,K1-1b7-IFNβ-HSA-27 and K1-1b7-IFNβ-HSA-33.K1-1b7-IFNβ-HSA-26 and K1-1b7-IFNβ-HSA-27cells were selected for batch culture to detect the expression levels of IFNβ-HSA fusion proteins.The expression levels were 9.41 mg·L^-1 and 13.85 mg·L^-1 respectively.