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

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。

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 FUT8 and 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.