CRISPR Mediated Chromosome Labeling in Mouse Cells

Abstract: 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 sgRNA 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 piggyBac 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 Neuro-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 mESC， 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.

CSTR: 32200.14.cjcb.2018.02.0012