CRISPR系统介导的小鼠细胞染色体标记

CRISPR Mediated Chromosome Labeling in Mouse Cells

细胞基因组生物学功能的发挥依赖于其染色质空间组成方式和动态活动,而对染色体的动态变化进行研究的首要任务,就是通过一种简单而有效的方法实现基因组中特定序列的标记。近年来,随着CRISPR技术的发展,应用其对特定基因进行标记将成为研究染色体动态变化的有力手段。该研究中,对CRISPR系统的sgRNA支架序列进行F+E修饰,增强了dCas9蛋白复合体对DNA的结合能力,获得支架序列增强型CRISPR(enhance sgRNA scaffold-CRISPR,Esgs-CRISPR)系统。接着,进一步将Esgs-CRISPR标记系统与PB转座系统以及Tet-on系统相结合,构建了适用于稳定标记细胞的质粒系统,即PB-Tet-on-Esgs-CRISPR(PTE-CRISPR)系统。在PTE-CRISPR质粒中插入特定的sgRNA序列,通过脂质体转染成功标记了小鼠神经瘤母细胞(mouse neuroblastoma cell line-2A,N2A)的端粒和卫星序列。通过与表达转座酶(PB transposase,PBase)的质粒共转染小鼠胚胎干细胞(mouse embryonic stem cells,mESC),该研究成功标记了mESC的端粒及卫星序列,并通过流式细胞术筛选获得了标记端粒和卫星序列的稳转细胞系。该研究实现了CRISPR系统介导的mESC染色体特定基因序列的标记,为进一步研究活细胞内染色体结构和动态变化提供了基础。

The biological function of genome bases on its spatial organization and dynamic in different situations. The primary task of studying the spatial dynamics of chromosome is to achieve a simple and effective method of labeling specific genomic sequence. With the development of CRISPR, this technology has provided a powerful labeling means for imaging of specific genomic locus in chromosome. In this study, the scaffold of sg RNA had been F＋E modified to enhance the binding capacity between dCas9 and DNA sequence, and received enhance sgRNA scaffold-CRISPR（Esgs-CRISPR） system. Furthermore, this Esgs-CRISPR labeling system was combind with piggy Bac transposition system and Tet-on system, to construct a PB-Tet-on-Esgs-CRISPR（PTE-CRISPR） system, which was suitable to build a stable labelled cell line. By inserting a particular sgRNA in the PTE-CRISPR plasmid and transfecting it into mouse neuroblastoma cell line-2A（N2A）, we successfully imaged the telomere and major satellite genomic locus in N2A cells. To establish a stable labelled mouse embryonic stem cells（mESC）, we cotransfected the sgRNA-PTE-CRISPR plasmid with a PBase expressing plasmid into m ESC, and we obtained cell lines with stably labeled telomere and major satellite by fluorescent activated cell sorting. In this study, we showed that the CRISPR system enables special genome locus labeling in mESC, which provided a robust tool for further research on the organization and dynamic of chromosome in living cells.