The ability to genetically manipulate microorganisms has been essential for understanding their biology and metabolism. Targeted genome editing relies on highly efficient homologous recombination, and while this is readily observed in the yeast Saccharomyces cerevisiae, most non-conventional yeast species do not display this trait and remain recalcitrant to targeted editing methods. CRISPR-based editing can bypass the requirement for high levels of native homologous recombination, enabling targeted modification to be more broadly implemented. While genetic transformation has been reported previously in Brettanomyces bruxellensis, a yeast with broad biotechnological potential and responsible for significant economic losses during the production of fermented beverages, targeted editing approaches have not been reported. Here, we describe the use of an expression-free CRISPR-Cas9 system, in combination with gene transformation cassettes tailored for B. bruxellensis, to provide the means for targeted gene deletion in this species. Deletion efficiency was shown to be dependent on homologous flanking DNA length, with higher targeting efficiencies observed with cassettes containing longer flanking regions. In a diploid strain, it was not possible to delete multiple alleles in one step, with heterozygous deletants only obtained when using DNA cassettes with long flanking regions. However, stepwise transformations (using two different marker genes) were successfully used to delete both wild-type alleles. Thus, the approach reported here will be crucial to understand the complex physiology of B. bruxellensis.
• The use of CRISPR-Cas9 enables targeted gene deletion in Brettanomyces bruxellensis.
• Homozygous diploid deletions are possible with step-wise transformations.
• Deletion of SSU1 confirmed the role of this gene in sulphite tolerance.
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All strains and plasmids used in this study are available from the Australian Wine Research Institute Wine Microorganism Culture Collection. Sequencing reads included in this study and the reference genome of AWRI1613 are available in NCBI under Bioproject PRJNA622385.
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The AWRI is supported by Australia’s grape growers and winemakers through their investment body Wine Australia with matching funds from the Australian Government. The AWRI and the University of Adelaide are members of the Wine Innovation Cluster in Adelaide.
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Varela, C., Bartel, C., Onetto, C. et al. Targeted gene deletion in Brettanomyces bruxellensis with an expression-free CRISPR-Cas9 system. Appl Microbiol Biotechnol (2020). https://doi.org/10.1007/s00253-020-10750-5
- Gene deletion