利用CRISPR/CAS9技术构建内源WDR82标签蛋白融合表达的小鼠胚胎干细胞系

Objective: In this study, CRISPR/Cas9 technique was used to insert Flag or TAP-Flag into the terminal of wdr82 gene to generate embryonic stem cells with endogenous WDR82 protein fused with two different tags. Methods: First, sgRNA pairs were designed near the termination codon of WDR82 protein. At the same time, targeting plasmids were constructed, which contain Flag or TAP-Flag tag, left and right homologous arms with the length of 500bp-1kb. Second, Cas9 plasmid, sgRNA plasmid and targeting plasmid were co-transfected into mESCs. After Neomycin selection, single colony was picked, subjected to genotyping and sequencing to screen the cell lines with endogenous WDR82 protein fused with Flag or TAP-Flag. Third, Western Blot and Co-IP experiments were performed to examine the endogenous expression of WDR82 Flag/TAP-Flag fusion protein and its interaction with SETD1A/SETD1B subunits. Results: The knock-in efficiency of Flag and TAP-Flag targeting was 11.46% and 18.75%, respectively. The expressed WDR82-FLAG/TAP-FLAG fusion proteins can interact with SETD1A and ASH2L protein.