A modular cloning toolbox including CRISPRi for the engineering of the human fungal pathogen and biotechnology hostCandida glabrata

ABSTRACT The yeast Candida glabrata is an emerging, often drug-resistant opportunistic human pathogen, that can cause severe systemic infections in immunocompromised individuals. At the same time, it is a valuable biotechnology host that naturally accumulates high levels of pyruvate – a valuable chemical precursor. Tools for the facile engineering of this yeast could greatly accelerate studies on its pathogenicity and its optimization for biotechnology. While a few tools for plasmid-based expression and CRISPR/Cas-based genome engineering have been developed, there is no well-characterized cloning toolkit available that would allow the assembly of pathways or genetic circuits in a modular fashion. Here, by re-using and characterizing the Saccharomyces cerevisiae -based yeast molecular cloning toolkit (YTK) in C. glabrata and by adding missing components, we build a well-characterized CgTK ( Candida glabrata toolkit). We used the CgTK to build a CRISPR interference system for C. glabrata that can be used to generate selectable phenotypes via single-gRNA targeting such as required for genome-wide library screens.