Rapid fabrication of designer genome-wide yeast libraries

快速构建设计者全基因组酵母文库

• 批准号: 2273720
• 金额: --
• 依托单位: Queen Mary University of London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2273720
• 关键词: Rapid; fabrication; designer; genome; wide

Aims: We propose to create a system (Rapid Tag Switching or RTS) to enable researchers to create custom libraries of yeast strains, each strain encoding a different tagged protein, in as little as one week. Such libraries could encode any genetically-encoded tag conceivable. Examples could include 'switchable' fluorophores for high-throughput super-resolution imaging, or conditional degrons, which would facilitate study of essential proteins across the genome - the possibilities are endless. For our own studies on the kinetochore, we wish to define how specific genetic changes impact upon the process of segregating chromosomes during cell division, since mis-segregated chromosomes are a hallmark of cancer cells. We will use RTS to create libraries of strains encoding novel fluorophores, both for multi-channel imaging and super-resolution imaging that will allow us to quantitatively measure such changes and map the position of kinetochore regulators. Additionally, we would aim to create a library of strains where each protein can be conditionally degraded or 'knocked sideways' (removed to a specific location within the cell). These libraries would be used to create specific alterations that result in chromosome mis-segregation and define how these changes affect cells - essentially modelling the changes seen in cancer cells.Methods: We will combine three existing tools to create an effective method of RTS. First, we will make use of an existing and well-characterised yeast GFP library in which each gene is fused with the open reading frame encoding GFP. Second, CRISPR-Cas9 mediated cleavage of the sequence encoding GFP will greatly enhance genetic recombination with a homologous fragment of DNA encoding the new tag of choice. Third, both the Cas9 gene (plus RNA guide) and the homologous fragment will be delivered using a mating-based plasmid-transfer method called Selective Ploidy Ablation (SPA). The SPA method allows DNA constructs to be transferred by copying yeast strains together on agar plates; a huge cost and time saving over using traditional transformation protocols. A key step in streamlining this process will be to 2 optimise colony transfers using a high-throughput pinning robot (ROTOR, Singer Instruments Ltd) to allow an entire yeast genome-wide library (~6000 strains) to be copied on a single plate. The newly-tagged strains will contain a selectable genetic marker to ensure the library strains have been converted.Summary: There have been several attempts to achieve a system in yeast that can rapidly create bespoke libraries. However, these typically depend upon a specific starting library (e.g. SWAP-TAG) and use sporulation as an intermediate step, which adds considerable time to the proceedure. The RTS can utilise any genome-wide library as a starting point and can be achieved in around a week. Thus this system brings the ability to create bespoke libraries within the reach of most microbial laboratories. The ability of researchers to rapidly and cheaply create custom libraries encoding tagged proteins would be immensely useful for researchers in many fields.

目的：我们建议创建一个系统（快速标签切换或RTS），使研究人员能够在短短一周内创建酵母菌株的定制库，每个菌株编码不同的标记蛋白。这样的库可以编码任何可能的基因编码标签。例如，用于高通量超分辨率成像的“可切换”荧光团，或有助于研究整个基因组中必需蛋白质的条件退化——可能性是无穷无尽的。对于我们自己对着丝点的研究，我们希望确定特定的遗传变化如何影响细胞分裂过程中染色体的分离过程，因为错误分离的染色体是癌细胞的一个标志。我们将使用RTS创建编码新型荧光团的菌株库，用于多通道成像和超分辨率成像，这将使我们能够定量测量这些变化并绘制着丝点调节体的位置。此外，我们的目标是创建一个菌株库，其中每个蛋白质都可以有条件地降解或“侧敲”（移到细胞内的特定位置）。这些文库将被用来创造导致染色体错误分离的特定改变，并定义这些改变如何影响细胞——本质上是模拟在癌细胞中看到的变化。方法：我们将结合三种现有的工具来创造一种有效的RTS方法。首先，我们将利用一个现有的和特性良好的酵母GFP文库，其中每个基因与编码GFP的开放阅读框融合。其次，CRISPR-Cas9介导的编码GFP序列的切割将极大地增强与编码新标签的同源DNA片段的遗传重组。第三，Cas9基因（加上RNA引导）和同源片段将使用一种基于配对的质粒转移方法，称为选择性倍性消融（SPA）。SPA方法允许通过在琼脂板上复制酵母菌株来转移DNA结构；与使用传统转换协议相比，节省了大量的成本和时间。简化这一过程的关键步骤将是使用高通量钉钉机器人（ROTOR, Singer Instruments Ltd）优化菌落转移，以便在单个板上复制整个酵母全基因组文库（约6000株）。新标记的菌株将包含一个可选择的遗传标记，以确保文库菌株已被转化。总结：已经有几次尝试在酵母中实现一个可以快速创建定制库的系统。然而，这些通常依赖于特定的起始库（例如SWAP-TAG），并使用孢子形成作为中间步骤，这增加了相当多的时间。RTS可以利用任何全基因组文库作为起点，并且可以在大约一周内完成。因此，该系统带来了在大多数微生物实验室范围内创建定制库的能力。研究人员能够快速而廉价地创建编码标记蛋白质的定制库，这对许多领域的研究人员来说都是非常有用的。