基于细胞融合开发双基因共敲除技术

Development of a Technique for Double Gene Knockout Using Cell Fusion

摘要:

基因编码工具（例如CRISPR/Cas9）的出现使敲除哺乳动物细胞基因成为现实。然而，实现细胞的多基因敲除成功率低且所需时间长。细胞融合技术是构建杂合细胞的常用方法，通过融合两种不同表型的细胞，构建出一种具有杂合表型的新型细胞。但是，由于基因的互补作用，通过基因敲除而获得的性状在细胞融合后成为隐性性状，融合细胞不能表现该性状。本研究的目的是设计一种通过细胞融合技术和CRISPR/Cas9技术获得基因双敲除细胞的方法。由于现阶段没有关于HEK 293细胞株细胞融合的参考数据，所以首先在HEK 293野生型细胞株中分别敲除GPI生物合成必需的PIGA或PIGK，获得PIGA-KO细胞株和PIGK-KO细胞株，并以这两个细胞株作为模型进行条件优化。经过反应条件优化，HEK 293细胞的融合效率显著提高，且实现了FUT8敲除细胞株和ST6GAL1敲除细胞株的快速融合；其次是将细胞融合技术与CRISPR/Cas9技术结合从而实现多种糖基因的快速敲除。将分别含有FUT8和ST6GAL1目标导向RNAs且都能稳定表达Cas9蛋白的两个细胞株进行融合，即可获得FUT8和ST6GAL1基因都被敲除的双敲细胞株。实验结果表明，将细胞融合技术和CRISPR/Cas9技术结合可简便而快速地获得基因双敲除细胞株。

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.