CATS: Cas9-assisted tag switching. A high-throughput method for exchanging genomic peptide tags in yeast

Abstract

Abstract Background 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. 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. Results We 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. Conclusions The 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.

Keywords

• CRISPR-Cas9
• Yeast
• Array
• GFP collection
• SPA
• Tag switching