CRISPR-mediated chromosome-specific labelling for live cell imaging applications

基因组携带着构建和调节有机体的信息。它们是由DNA组成的复杂的物理结构，在整个细胞生命周期中经历戏剧性的动态变化。在人类中，DNA被包装成23个离散的结构，称为染色体。几十年来，人们已经在活细胞中使用光学和荧光显微镜对它们进行了研究。然而，在这些技术中，所有的染色体都无法相互区分。这种局限性阻碍了对染色体生物学中许多问题的研究。在这项建议中，我们的目标是开发一系列适合活细胞研究的细胞系，每个细胞系都有一个已知的染色体被荧光标记。为了实现这一点，我们将使用最近开发的CRISPR方法的修改，该方法允许通过引入特定的RNA片段将名为Cas9的DNA切割酶靶向基因组上的任何位置。我们将使用修改版本的Cas9，其中切割活动已被禁用，因此它仍然粘在基因组上选定的位置。通过将荧光蛋白附着到改良的Cas9上，并将许多副本定向到选定染色体上的特定位置，我们将构建一个具有与最先进的活细胞成像兼容的独特染色体识别符的“条形码”系统。这项技术将开辟新的研究途径，例如染色体如何在细胞核中排列，染色体是如何包装的，以及它们在细胞分裂过程中是如何分离的。

Genomes carry the information to build and regulate organisms. They are elaborate physical structures comprising DNA that undergoes dramatic dynamic changes throughout the cell lifecycle. In humans, the DNA is packaged into 23 discrete structures called chromosomes. These have been studied for many decades in live cells using light and fluorescence microscopy. However, in these techniques all of the chromosomes are indistinguishable from each other. This limitation has held back the study of many problems in chromosome biology. In this proposal, we aim to develop a range of cell lines suitable for live cell studies, each with a single known chromosome fluorescently labelled. To accomplish this, we will use a modification of the recently developed CRISPR method which allows the targeting of a DNA cutting enzyme called Cas9 to anywhere on the genome by introducing a specific piece of RNA. We will use a modified version of Cas9 where the cutting activity has been disabled such that it remains stuck to the chosen location on the genome. By attaching fluorescent proteins to the modified Cas9 and directing many copies to a specific location on a chosen chromosome we will construct a 'barcoding' system with unique chromosome identifiers compatible with state-of-the-art live cell imaging. This technology will open up new avenues of research into, for example, how chromosomes are arranged in the nucleus, how chromosomes are packaged and how they are segregated during cell division.