Knockout Of DNMT3a Gene In Both CHO And HEK293T Cells As Well As Targeted Integration Of CTLA4Ig Gene In CHO Cells Via Crispr/Cas9 Technology

Background and Purpose:Crispr,short for Clustered Regularly Interspaced Short Palindrome Repeats,was originally found in the genome of a number of archaea and bacterials,is considered to be relevant to immune responses of these prokaryotes.With the research about it going deeper,Crispr/Cas9 has been adapted to a popular gene editing technology gradually.Compared with previously developed genome engineering technologies such as zinc finger nucleases(ZFNs)and transcription activator-like effector nucleases(TALENs),Crispr/Cas9 system is easier to use,less time-consuming,cost-effective,as well as with a higher editing efficiency.In our research,Crispr/Cas9 system was successfully employed to targeted engineer the genome of two most widely used cell lines for biopharmaceuticals: Chinese Hamster Ovary(CHO)cells and Human Embryo Kidney(HEK293T)cells.Currently more than a half of the therapeutic recombinant proteins approved on market are produced in mammalian cells,however,there is a major drawback with the mammalian cell expression system.It is the instability of recombinant cell lines.With the culture time extending,the productivity of the cell line will drop significantly.Some evidence shows that this instability may be related with epigenetic modifications,especially DNA methylation.So far only three classes of enzymes responsible for DNA methylation are found in mammalian cells.And one of the three enzymes,DNA methyltransferases 3a(DNMT3a)was chose by us for further study.The gene encoding this enzyme was first disrupted with Crispr/Cas9 system and then alterations were observed before and after the gene was knockout.And we mainly investigated whether the productivity of exogenous proteins was elevated when the gene was disrupted.At the meantime,recombinant cell lines were constructed mainly by random integration at present.But random integration will give rise to unwanted problems due to lack of control of the gene insertion-termed the “position effect”.In order to circumvent such a situation,scholars are focusing on targeted integration.Since Crispr/Cas9 system has unique advantages in gene editing,a variety of researchers are using this technology to insert the gene of interest(GOI)into the specific locus in the genome.In our research,we utilized the Crispr technology to targeted integrate the cytotoxic T Lymphocyte Antigen 4(CTLA4Ig)gene into the fucosyltransferases 8(FUT8)locus in CHO cells.Methods:Disruption of DNMT3 a gene in both CHO cells and HEK293 T cells: Two pairs of sgRNA targeting the key functional regions of DNMT3 a protein were designed and constructed.Then the two pairs of sgRNA were co-transfected into CHO cells,following limiting dilution to plate transfected cells into 96 well-plates to obtain single cell clones.Green fluorescence protein(GFP)was used as the model protein to assess whether the productivity of transgene was improved or not when the DNMT3 a gene was disrupted.Targeted integration of CTLA4 Ig gene into the FUT8 locus of CHO cells: A total of five pairs of sgRNA targeting the sites around the start codon ATG were designed and their indel efficiency was evaluated.The two most efficient sgRNA pairs were chose for further use.The two picked sgRNA pairs together with the donor plasmid were co-transfected into CHO cells.Transfected cells were subjected to limiting dilution to obtain single cell clones followed by a drug-selection process.Single cell clones were picked up and detected to obtain correctly targeted cell lines.Results:Disruption of DNMT3 a gene in both CHO cells and HEK293 T cells: Finally one CHO cell line with DNMT3 a gene completely disrupted(referred to as DNMT3a-CHO)as well as one HEK293 T cell line(referred to as DNMT3a-HEK293T)with DNMT3 a gene completely disrupted was obtained through limiting dilution.UMS was applied to analyze the methylated cytosine content present in the whole genome.And results showed that the methylated cytosine in DNMT3a-CHO genome decreased by about 27% compared with that of wild type CHO(referred to as WT-CHO).While in DNMT3a-HEK293 T genome,methylated cytosine decreased by approximate 22% compared to wild type HEK293T(referred to as WT-HEK293T).As for growth profile,there is no difference between WT-CHO and DNMT3a-CHO cell line,however,DNMT3a-HEK293 T cell line grows much slower than WT-HEK293 T cell line.When GFP was applied as the model protein to evaluate whether transgene in DNMT3 a disrupted cell line is improved,results demonstrated that the productivity was not affected for CHO cell line while in HEK293 T cell line,loss of DNMT3 a gene caused great loss of GFP.Targeted integration of CTLA4 Ig gene into the FUT8 locus of CHO cells: The editing efficiency of the designed five pairs of sgRNA ranged from 17.7% to 31.7%.The most efficient sgRNA upstream or downstream of the start codon ATG were picked up for transfection.56 single cell clones were finally obtained after drug was applied into the cells transfected with sgRNA and donor plasmid.19 cell lines of the 56 single cell clones were identified as correctly targeted events after detection with polymerase chain reaction(PCR)and Western Blot.The targeting efficiency reached up to 33.9%.