Temporal manipulation of genetic circuits in single cells

单细胞遗传电路的时间操纵

• 批准号: BB/P027040/1
• 负责人: Michael White
• 金额: $ 15.55万
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FP027040%2F1
• 关键词: Temporal; manipulation; genetic; circuits; single

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.

生物成像是现代细胞生物学的主要工具。一个关键特征是开发了活细胞成像，用于跟踪和测量细胞过程，如细胞分裂、细胞死亡和从细胞外部传输信号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质：荧光蛋白和荧光蛋白。为了理解复杂的细胞系统，拥有特定的操作技术也很重要，这些技术可以以明确定义的方式扰乱关键过程。目前的干扰技术涉及突变或阻断基因活性，然而这些是不可控制或可逆的。一项重要的新技术已经从细菌中出现，称为CRISPR。它是一种编辑基因的天然细菌防御系统。该系统的核心是一种蛋白质Cas9，当被引导RNA靶向时，它可以有效地编辑基因，将其活性引导到特定基因，并可以整合和指导特定的DNA序列变化。最近，已经开发出了Cas9的新突变版本，可以在不改变基因序列的情况下抑制或激活基因。我们现在将使用一种已被分成两部分的Cas9蛋白。它可以与光敏蛋白质融合，当你照射它们时，它们就会结合在一起。因此，功能性Cas9仅在细胞被照射时形成。我们的目标是利用这种方法来开发新的方法来编辑，抑制和激活基因以响应光照。这使得基因扰动得到精确的定时控制，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途，并可以在生物学和医学中有更广泛的应用。

项目成果

Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation

• DOI: 10.1186/s13059-019-1776-2
• 发表时间: 2019-08-26
• 期刊: GENOME BIOLOGY
• 影响因子: 12.3
• 作者: Gurumurthy, Channabasavaiah B.;O'Brien, Aidan R.;Burgio, Gaetan
• 通讯作者: Burgio, Gaetan