Zhang Shuxian, Song Zhan
College of Marines Life Science, Ocean University of China, No. 5 Yushan Road, Qingdao, 266003, Shandong, People's Republic of China.
J Mol Histol. 2017 Aug;48(4):293-299. doi: 10.1007/s10735-017-9727-2. Epub 2017 May 25.
The biological function of chromatin bases on its spatial organization and dynamic activities in different situations. Labeling and tracing of genomic sequences has been a huge challenge in studying the spatial dynamics of chromatin. We reported the development of 'all-in-one Casilio system (Aio-Casilio)', a new system that enables the labeling of endogenous genomic loci. The Aio-Casilio system consists of the dCas9 protein, mClover fused with the Pumilio and FBF proteins RNA-binding domain (PUF domain) and an U6-sgRNA appended with multiple PUF-binding site(s). Here we showed that the Aio-Casilio system is robust tool for imaging of repetitive elements in telomeres and major satellite in N2A cells. Furthermore, we developed a PBTon-Aio-Casilio System, which enables the visualization of repetitive sequences in mES cells. However, this system failed to establish a labeled ES cell line when we attempted to establish a stable labeling cell line for living cell image. This Aio-Casilio imaging tool has potential to significantly improve the capacity to study the conformation of chromosomes in living cells.
染色质的生物学功能基于其在不同情况下的空间组织和动态活动。基因组序列的标记和追踪一直是研究染色质空间动态的巨大挑战。我们报道了“一体化卡西利奥系统（Aio-Casilio）”的开发，这是一种能够对内源基因组位点进行标记的新系统。Aio-Casilio系统由dCas9蛋白、与Pumilio和FBF蛋白RNA结合结构域（PUF结构域）融合的mClover以及附加有多个PUF结合位点的U6-sgRNA组成。在这里，我们表明Aio-Casilio系统是用于对N2A细胞端粒中的重复元件和主要卫星进行成像的强大工具。此外，我们开发了PBTon-Aio-Casilio系统，该系统能够在mES细胞中可视化重复序列。然而，当我们试图建立用于活细胞成像的稳定标记细胞系时，该系统未能建立标记的ES细胞系。这种Aio-Casilio成像工具具有显著提高研究活细胞中染色体构象能力的潜力。
