Development Of A Technique For Double Gene Knockout Using Cell Fusion

Knockout and insertion of gene and regulation of gene expression in mammalian cells can be achieved with the help of gene-editing tools,such as zinc finger structure,transcriptional activator like effectors and CRISPR/Cas9 technology.However,although zinc finger structures and transcriptional activator like effectors have begun to achieve targeted gene editing,both of them are costly and difficult to be obtained.In contrast,the CRISPR/Cas9 technology,developed on the principle of Cas9 protein-mediated DNA double-strand breakage,CRISPR/Cas9 technology can edit gene more precisely,and shortens the gene editing period,reduces the workload and reduces the cost effectively.Based on the above situation,in thie study,we constructed several HEK 293 derived cell lines with the help of CRISPR/Cas9 technology,such as PIGA-KO-Hyg-GFP cell line,PIGK-KO-BSD-BFP cell line,FUT8-KO-Hyg cell line,ST6GAL1-KO-BSD cell line,FUT8-cr1-KO-Hyg cell line and ST6GAL1-cr1-KO-BSD cell line.These cell lines provided experimental models for subsequent studies.It is well known that two haploid yeast cells that have been knockout two different genes can produce double knockout yeast cells through match and spore formation.So,is it possible for mammalian cells to knock out target genes by CRISPR/Cas9 technology and construct gene double knockout cell lines by the way that construct gene double knockout diploid yeast cells? Biologists have known for years that different species cells can be fused and produce hybrid cells,resulting in cell lines with several phenotypes.In other words,the new cell lines can also be obtained by existing gene knockout cells through cell fusion technology directly,and without the complicated and lengthy work of gene editing.And this is the advantage of cell fusion.Chinese hamster ovary cells and hela cells are used as normal models for cell fusion,but it’s more advantageous to use human embryonic kidney cells 293(HEK 293)as cell fusion model for the prevention and treatment of human diseases.In this study,HEK 293 PIGA-KO-Hyg-GFP cell line and HEK 293PIGK-KO-BSD-BFP cell line were used as models to construct HEK 293 PIGA-KO-Hyg-GFP &PIGK-KO-BSD-BFP fused cell line.The condition to do cell fusion was optimized and then improved the efficiency of cell fusion and constructed HEK 293 FUT8-KO-Hyg & ST6GAL1-KOBSD fused cell line by the optimized method using the HEK 293 FUT8-KO-Hyg cell line and HEK293 ST6GAL1-KO-BSD cell line.The experiments of this study proved that HEK 293 cells can be fused and the fusion cell lines were healthy and stable.For some scientific or production requirements,it’s necessary to construct cell lines rapidly that knockout multiple genes simultaneously.But until now the relevant research has not achieved satisfactory results.In addition,cell fusion technology is a quick way to obtain cell lines that with double dominant traits,for example,the HEK 293 PIGA-KO-Hyg-GFP & PIGK-KO-BSD-BFP fused cell line can expressing both green fluorescent proteins and blue fluorescent proteins,but cannot obtain double invisible traits,for example,PIGA-PIGK-DKO cell line.When two kinds of cells that with different knockout genes are fused,the traits that acquired by the original cell due to gene knocking become to be invisible straits and cannot be detected anymore,because of the complementary effect of genes.Therefore,this study designed a method to obtain gene double knockout cells through existing gene knockout cells,it means that,combined with cell fusion and CRISPR/Cas9 technology to obtain double knockout HEK 293 derived cell lines.The FUT8-ST6GAL1-DKO cell line was constructed by this from HEK 293 FUT8-cr1-KO-BSD cell line and HEK 293 ST6GAL1-cr1-KO-Puro cell line.This study combines cell fusion and CRISPR/Cas9 technology successfully and constructed gene double knockout HEK 293 derived cell line,its significance in the development of research and industrial production can be summarized as follows:(1)got fused cell line using HEK 293 cell line successfully,and improved the cell fusion efficiency to 8.8%,this result provides data reference for the cell fusion research using HEK 293 cell line;(2)developed a new method for constructing double-gene knockout cells by combining cell fusion and CRISPR/Cas9 technology,which can construct multi-gene knockout cells directly on the basis of existing gene knockout cell lines;(3)the combination of cell fusion and CRISPR/Cas9 technology shortens the time required to obtain cell lines effectively with multiple gene knocked out at the same time,which has great significance for the rapid development of scientific research and efficient production of industrial products.