Abstract
BackgroundThe creation of arrays of yeast strains each encoding a different protein with constant tags is a powerful method for understanding how genes and their proteins control cell function. As genetic tools become more sophisticated there is a need to create custom libraries encoding proteins fused with specialised tags to query gene function. These include protein tags that enable a multitude of added functionality, such as conditional degradation, fluorescent labelling, relocalization or activation and also DNA and RNA tags that enable barcoding of genes or their mRNA products. Tools for making new libraries or modifying existing ones are becoming available, but are often limited by the number of strains they can be realistically applied to or by the need for a particular starting library.ResultsWe present a new recombination-based method, CATS – Cas9-Assisted Tag Switching, that switches tags in any existing library of yeast strains. This method employs the reprogrammable RNA guided nuclease, Cas9, to both introduce endogenous double strand breaks into the genome as well as liberating a linear DNA template molecule from a plasmid. It exploits the relatively high efficiency of homologous recombination in budding yeast compared with non-homologous end joining.ConclusionsThe method takes less than 2 weeks, is cost effective and can simultaneously introduce multiple genetic changes, thus providing a rapid, genome-wide approach to genetic modification.
Highlights
The creation of arrays of yeast strains each encoding a different protein with constant tags is a powerful method for understanding how genes and their proteins control cell function
Testing plasmid loss and efficiency of Cas9 cleavage in W303 yeast Homologous recombination at a given locus is greatly facilitated by the presence of a Double strand break (DSB) [37], since endogenous repair mechanisms are acting directly on the genome
After following the indicated protocol (Fig. 4a), we found that only the active endonuclease plasmid resulted in G418 and 5-fluoroorotic acid (5-FOA) resistant colonies and fluorescence imaging revealed that these strains expressed Red Fluorescent Protein (RFP) tagged Htb2 and Rpa29 (Supplementary Table 3)
Summary
The creation of arrays of yeast strains each encoding a different protein with constant tags is a powerful method for understanding how genes and their proteins control cell function. As genetic tools become more sophisticated there is a need to create custom libraries encoding proteins fused with specialised tags to query gene function. A number of subsequent budding yeast collections have been made, including a set of GFP tagged strains to determine the localization of each cellular protein [19] and a dual epitope tag (Tandem affinity purification or TAP) collection for analysing protein levels and protein-protein interactions [11]. All of these collections have been widely used and provided important information on the function of eukaryotic cells
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