Abstract:Emerging of genome-editing tools such as CRISPR/Cas9 enables us to perform gene knockout (KO) in mammalian cells. However, KO of multiple genes in a cell is inefficient and time consuming. Cell fusion, frequently used for hybridoma construction, is a way to fuse two different cells and create a new cell line with hybrid phenotypes. However, the recessive phenotype by gene-KO cannot be obtained by cell fusion, because gene complementation occurs from the other cell to be fused. The purpose of this study is to develop a method to obtain double KO cells using cell fusion combined with CRISPR/Cas9. The process and method are as follows. Without available cell fusion data using HEK 293. We first knocked out PIGA or PIGK which were necessary for GPI biosynthesis, in HEK 293 wild-type cell line to obtain PIGA-KO cell line and PIGK-KO cell line. Then the two cell lines were as optimized as models. By optimizing the fusion condition, the fusion efficiency of HEK 293 cells was greatly improved. Secondly, we combined cell fusion technology with CRISPR/Cas9 technology to achieve the knockout of multiple sugar genes. Under optimized condition, fusion of FUT8-KO cells and ST6GAL1-KO cells was performed. Both FUT8and ST6GAL1 were successfully disrupted after cell fusion. These results suggest a simple and quick method to acquire double KO cells using our developed method combined cell fusion with CRISPR/Cas9.
