A new inducible CRISPR-Cas9 system useful for genome editing and study of double-strand break repair in Candida glabrata.

Abstract

In recent years, the CRISPR-Cas9 system has proven extremely useful for genome editing in many species, including the model yeast Saccharomyces cerevisiae and other yeast species such as Candida glabrata. Inducible CRISPR-Cas9 systems have the additional advantage of allowing to separate the transformation step of the organism by the CRISPR-Cas9 system, from the cutting and repair steps. This has indeed been developed in S.cerevisiae, where most inducible expression systems rely on the GAL promoters. Unfortunately, C.glabrata is gal- and lacks the GAL genes, like many other yeast species. We report here the use of a vector expressing cas9 under the control of the MET3 promoter, with the guide RNA cloned into the same plasmid. We show that it can be used efficiently in C.glabrata, for both described outcomes of CRISPR-Cas9-induced chromosome breaks; nonhomologous end joining in the absence of a homologous repair template; and homologous recombination in the presence of such a template. This system therefore allows easy editing of the genome of C.glabrata, and its inducibility may allow identification of essential genes in this asexual yeast, where spore lethality cannot be observed, as well as the study of double-strand break repair.