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The CRISPR/Cas9 technology has greatly improved genome editing in  Saccharomyces cerevisiae  over recent years. However, several current CRISPR/Cas9 systems suffer from work-intensive cloning procedures and/or the requirement of co-transforming target cells with multiple system components simultaneously which can reduce the effectivity of such applications. Here, we present a new set of all-in-one CRISPR/Cas9 vectors that combine unique benefits of different already existent systems in order to further expand the technology’s design possibilities. Our vectors mediate constitutive gRNA expression whereas Cas9 expression is either driven from a constitutive or an inducible promoter. The introduction of desired gRNA targeting sequences into our inducible single gRNA vector relies just on  in vivo  homologous recombination-mediated assembly of overlapping single-stranded oligonucleotides, thus reducing efforts of plasmid cloning to an absolute minimum. By employing the inducible system, yeast cells can be easily preloaded with plasmids encoding for a functional CRISPR/Cas9 system, thereby chronologically separating the cloning procedure from the genome editing step. Gene knockouts could be achieved with high efficiency and effectivity by simply transforming preloaded cells with a selectable disruption cassette without the need of co-introducing any CRISPR/Cas9 system component. We also show the feasibility of efficient gene knockouts even when multiple gene copies were present such as in non-haploid strain backgrounds as well as the simultaneous deletion of two different genes in a haploid genetic background by using a multiplex variant of our inducible vector. The versatile applicability of our inducible vector system was further demonstrated by CRISPR/Cas9-mediated mating type switching of yeast.

Preloading budding yeast with all-in-one CRISPR/Cas9 vectors for easy and high-efficient genome editing