Temporal manipulation of genetic circuits in single cells

生物成像是现代细胞生物学的重要工具。一个关键特征是活细胞成像的发展，用于跟踪和测量细胞过程，如细胞分裂、细胞死亡和信号从细胞外传递以控制细胞行为的机制。支持这些发展的一项主要技术是使用天然发光蛋白：荧光素酶和荧光蛋白。为了理解复杂的细胞系统，拥有能够以明确定义的方式干扰关键过程的特定操作技术也很重要。目前的干扰技术涉及突变或阻断基因活性，但这些都是不可控制或可逆的。一项重要的新技术从细菌中出现，称为CRISPR。它是一种天然的细菌防御系统，可以编辑基因。该系统的核心是一种名为Cas9的蛋白质，当被引导rna靶向时，Cas9可以有效地编辑基因，将其活性导向特定基因，并可以整合和指导特定的DNA序列变化。最近，Cas9的新突变版本被开发出来，在不改变基因序列的情况下抑制或激活基因。我们现在将使用Cas9蛋白的一个版本，它已经被分成了两部分。这可以融合成光敏蛋白，当你照射它们时，它们会结合在一起。因此，只有当细胞被照亮时，才会形成功能性Cas9。我们的目标是利用这种方法开发新的方法来编辑、抑制和激活基因以响应光照。这提供了对基因扰动的精确定时控制，这是以前所缺乏的。它将允许更精确地研究基因功能，这是现代生物学中的一个关键问题。因此，这些工具将在生物成像实验中有很大的用处，并可以在生物学和医学上有更广泛的应用。

Bio-imaging is a major tool in modern cell biology. A key feature has been the development of live cell imaging for the tracking and measurement of cellular processes such as cell division, cell death and the mechanisms by which signals are transmitted from the outside of the cell to control cellular behaviour. One major technology that has underpinned these developments has been the use of natural light emitting proteins: luciferases and fluorescent proteins. In order to understand complex cell systems it is also important to have specific manipulation technologies that can perturb key processes in clearly defined ways. Current interference technologies involve mutating or blocking gene activity, however these are not controllable or reversible. A major new technology has emerged from bacteria called CRISPR. It is a natural bacterial defence system that edits genes. The core of this system is a protein, Cas9, which can efficiently edit genes when targeted by guide RNAs that direct its activity to specific genes, and can incorporate and direct specific DNA sequence changes. Recently, new mutated versions of Cas9 have been developed that repress or activate genes without changing their sequence. We will now use a version of the Cas9 protein that has been split into two pieces. This can be fused to light sensitive proteins that bind together when you shine light on them. As a result, functional Cas9 is formed only when the cell is illuminated. Our aim is to use this approach to develop new ways to edit, repress and activate genes in response to illumination. This gives precisely timed control of gene perturbation, something that has previously been lacking. It will allow more precise investigation of gene function, a critical issue in modern biology. These tools will therefore be of great use in bio-imaging experiments and can have broader applications in biology and medicine.