Rapid fabrication of designer genome-wide yeast libraries

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文库，每个菌株编码不同的标记蛋白。这样的文库可以编码任何可以想象到的遗传编码标签。例子可能包括用于高通量超分辨率成像的可切换的荧光团，或者条件退化，这将促进对整个基因组中必要蛋白质的研究--可能性是无穷的。对于我们自己对着丝粒的研究，我们希望确定特定的基因变化如何影响细胞分裂过程中的染色体分离，因为错误分离的染色体是癌细胞的标志。我们将使用RTS创建编码新荧光团的菌株文库，用于多通道成像和超分辨率成像，使我们能够定量测量这种变化并绘制动粒调节器的位置图。此外，我们的目标是建立一个菌株文库，其中每种蛋白质都可以有条件地降解或‘侧击’(移到细胞内的特定位置)。这些文库将被用来创建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-本质上模拟在癌细胞中看到的变化。方法：我们将结合三个现有的工具来创建一种有效的RTS方法。首先，我们将利用现有的和特征良好的酵母GFP文库，其中每个基因与编码GFP的开放阅读框架融合在一起。其次，CRISPR-Cas9介导的GFP编码序列的切割将极大地促进GFP与编码新标签的同源DNA片段的遗传重组。第三，Cas9基因(加RNA引导)和同源片段都将使用一种基于交配的质粒转移方法，称为选择性倍性消融(SPA)。SPA方法允许通过将酵母菌株复制到琼脂平板上来转移DNA结构；与使用传统的转化方案相比，节省了大量的成本和时间。简化这一过程的关键一步将是使用高通量钉扎机器人(ROTOR，Singer Instruments Ltd)优化菌落转移，以允许将整个酵母基因组文库(约6000株)复制到一个平板上。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。摘要：已经有几次尝试在酵母中实现一种系统，可以快速创建定制的文库。然而，这些通常依赖于特定的起始文库(例如交换标签)，并使用产孢子作为中间步骤，这增加了该过程相当长的时间。RTS可以利用任何全基因组文库作为起点，并可以在大约一周内实现。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图书馆的能力。研究人员能够快速而廉价地创建编码标记蛋白质的定制文库，这将对许多领域的研究人员非常有用。

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.