Rapid fabrication of designer genome-wide yeast libraries

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

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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.