Rapid and Efficient CRISPR/Cas9-Based Mating-Type Switching of <i>Saccharomyces cerevisiae</i>

Mating-type switching is a complex mechanism that promotes sexual reproduction in Saccharomycotina. In the model species Saccharomyces cerevisiae , mating-type switching is initiated by the Ho endonuclease that performs a site-specific double-strand break (DSB) at MAT , repaired by homologous recombination (HR) using one of the two silent mating-type loci, HMLalpha and HMRa . The reasons why all the elements of the mating-type switching system have been conserved in some Saccharomycotina, that do not show a sexual cycle nor mating-type switching, remain unknown. To gain insight on this phenomenon, we used the yeast Candida glabrata , phylogenetically close to S . cerevisiae , and for which no spontaneous and efficient mating-type switching has been observed. We have previously shown that expression of S . cerevisiae ’s Ho ( Sc Ho) gene triggers mating-type switching in C . glabrata , but this leads to massive cell death. In addition, we unexpectedly found, that not only MAT but also HML was cut in this species, suggesting the formation of multiple chromosomal DSBs upon HO induction. We now report that HMR is also cut by Sc Ho in wild-type strains of C . glabrata . To understand the link between mating-type switching and cell death in C . glabrata , we constructed strains mutated precisely at the Ho recognition sites. We find that even when HML and HMR are protected from the Ho-cut, introducing a DSB at MAT is sufficient to induce cell death, whereas one DSB at HML or HMR is not. We demonstrate that mating-type switching in C . glabrata can be triggered using CRISPR-Cas9, without high lethality. We also show that switching is Rad51-dependent, as in S . cerevisiae , but that donor preference is not conserved in C . glabrata . Altogether, these results suggest that a DSB at MAT can be repaired by HR in C . glabrata , but that repair is prevented by Sc Ho.

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“…This has been shown only recently in the model species S . cerevisiae [ 34 ]. No lethality is observed after a Cas9-DSB at MAT on plates.…”

Section: Discussionmentioning

confidence: 99%

A single Ho-induced double-strand break at the MAT locus is lethal in Candida glabrata

Maroc¹,

Zhou-Li²,

Boisnard³

et al. 2020

PLoS Genet

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“…Sexual reproduction is ubiquitous in eukaryotes and expands the genetic diversity by producing progeny that resemble their parents but are NOT identical to them [1]. The benefits of sexual reproduction include the purging of deleterious mutations from the genome.…”

Section: Introductionmentioning

confidence: 99%

Crossbreeding of Yeasts Domesticated for Fermentation: Infertility Challenges

Fukuda¹

2020

IJMS

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Sexual reproduction is almost a universal feature of eukaryotic organisms, which allows the reproduction of new organisms by combining the genetic information from two individuals of different sexes. Based on the mechanism of sexual reproduction, crossbreeding provides an attractive opportunity to improve the traits of animals, plants, and fungi. The budding yeast Saccharomyces cerevisiae has been widely utilized in fermentative production since ancient times. Currently it is still used for many essential biotechnological processes including the production of beer, wine, and biofuels. It is surprising that many yeast strains used in the industry exhibit low rates of sporulation resulting in limited crossbreeding efficiency. Here, I provide an overview of the recent findings about infertility challenges of yeasts domesticated for fermentation along with the progress in crossbreeding technologies. The aim of this review is to create an opportunity for future crossbreeding of yeasts used for fermentation.

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“…In yeasts, the system has been used, first and foremost, in S . cerevisiae , for genome engineering (DiCarlo et al, ) but also for the study of the repair of DSBs (Lemos et al, ) and for mating‐type switching (Xie et al, ). Several versions of the CRISPR‐Cas9 systems have also been designed for C .…”

Section: Introductionmentioning

confidence: 99%

“…In yeasts, the system has been used, first and foremost, in S. cerevisiae, for genome engineering (DiCarlo et al, 2013) but also for the study of the repair of DSBs (Lemos et al, 2018) and for mating-type switching (Xie et al, 2018). Several versions of the CRISPR-Cas9 systems have also been designed for C. glabrata (Cen, Timmermans, Souffriau, Thevelein, & Van Dijck, 2017;Enkler, Richer, Marchand, Ferrandon, & Jossinet, 2016;Vyas et al, 2018), with success in obtaining repair events of both NHEJ and HR types.…”

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confidence: 99%

“…The system relies on the sugar source in the medium, and these represent nontoxic and "physiological" growth conditions for the yeast, even though growth in glucose is always the most efficient one, all other conditions being equal. The GAL1 and GAL2 promoters have been used with CRISPR-Cas9 efficiently in S. cerevisiae (DiCarlo et al, 2013;Xie et al, 2018), but C. glabrata is gal − and lacks the GAL genes (Dujon et al, 2004), thereby prohibiting the use of these promoters. Luckily, another efficient inducible system for protein expression has been developed in C. glabrata that relies on the MET3 promoter (Zordan et al, 2013).…”

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confidence: 99%

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

Maroc

Fairhead

2019

Yeast

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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.

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Rapid and Efficient CRISPR/Cas9-Based Mating-Type Switching of Saccharomyces cerevisiae

Cited by 44 publications

References 28 publications

A single Ho-induced double-strand break at the MAT locus is lethal in Candida glabrata

A single Ho-induced double-strand break at the MAT locus is lethal in Candida glabrata

Crossbreeding of Yeasts Domesticated for Fermentation: Infertility Challenges

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

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