The yeast 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 genome engineering have been developed, there is no well-characterized cloning toolkit that would allow the modular assembly of pathways or genetic circuits. Here, by characterizing the -based yeast molecular cloning toolkit (YTK) in and by adding missing components, we build a well-characterized CgTK ( toolkit). We used the CgTK to build a CRISPR interference system for that can be used to generate selectable phenotypes via single-gRNA targeting such as is required for genome-wide library screens.