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自定义酵母菌株库，每个菌株编码不同的标记蛋白质。这样的文库可以编码任何可能的遗传编码标签。例子可能包括用于高通量超分辨率成像的“可切换”荧光团，或条件降解决定子，这将有助于研究整个基因组中的必需蛋白质-可能性是无限的。对于我们自己对动粒的研究，我们希望确定特定的遗传变化如何影响细胞分裂期间染色体分离的过程，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动粒调节器的位置。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“敲侧”（移到细胞内的特定位置）。这些库将被用来创建特定的改变，导致染色体错误分离，并定义这些变化如何影响细胞-基本上模拟在癌细胞中看到的变化。方法：我们将联合收割机三个现有的工具来创建一个有效的RTS方法。首先，我们将利用一个现有的和良好的特点，酵母GFP文库中的每个基因与开放的阅读框架编码GFP融合。其次，CRISPR-Cas9介导的编码GFP的序列的切割将极大地增强与编码所选新标签的DNA的同源片段的遗传重组。第三，Cas9基因（加上RNA向导）和同源片段将使用称为选择性倍性消融（SPA）的基于交配的质粒转移方法进行递送。SPA方法允许DNA构建体通过在琼脂平板上一起复制酵母菌株来转移;与使用传统转化方案相比，节省了巨大的成本和时间。简化该过程的关键步骤是使用高通量钉扎机器人（ROTOR，Singer Instruments Ltd）优化菌落转移，以允许在单个平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，以确保文库菌株已经被转换。总结：已经有几次尝试在酵母中实现一个系统，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SWAP-TAG），并使用孢子形成作为中间步骤，这增加了相当多的程序时间。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，该系统能够在大多数微生物实验室的范围内创建定制文库。研究人员能够快速、廉价地创建编码标记蛋白质的定制文库，这对许多领域的研究人员都非常有用。

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.