Fast and efficient CRISPR-mediated genome editing in Aureobasidium using Cas9 ribonucleoproteins

Kreuter, Johanna; Stark, Georg; Mach, Robert L.; Mach-Aigner, Astrid R.; Derntl, Christian

doi:10.1016/j.jbiotec.2022.03.017

Cited by 7 publications

(15 citation statements)

References 41 publications

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“…This should include both data-driven research, such as well-designed and carefully conducted transcriptomic experiments, and hypothesis-driven research that utilises the molecular tools that have become available in the two decades since the first systematic studies of halotolerance in these fungi. Among other tools, CRISPR-Cas9 is increasingly being used for the genetic manipulation of unconventional yeasts, including A. pullulans (Zhang et al 2019 ; Kreuter et al 2022 ), opening up new possibilities for deciphering the complex phenotype of halotolerant black yeasts.…”

Section: Genomics As the Future Of Black Yeast Researchmentioning

confidence: 99%

Black yeasts in hypersaline conditions

Gostinčar,

Gunde-Cimerman

2024

Appl Microbiol Biotechnol

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Extremotolerant and extremophilic fungi are an important part of microbial communities that thrive in extreme environments. Among them, the black yeasts are particularly adaptable. They use their melanized cell walls and versatile morphology, as well as a complex set of molecular adaptations, to survive in conditions that are lethal to most other species. In contrast to extremophilic bacteria and archaea, these fungi are typically extremotolerant rather than extremophilic and exhibit an unusually wide ecological amplitude. Some extremely halotolerant black yeasts can grow in near-saturated NaCl solutions, but can also grow on normal mycological media. They adapt to the low water activity caused by high salt concentrations by sensing their environment, balancing osmotic pressure by accumulating compatible solutes, removing toxic salt ions from the cell using membrane transporters, altering membrane composition and remodelling the highly melanized cell wall. As protection against extreme conditions, halotolerant black yeasts also develop different morphologies, from yeast-like to meristematic. Genomic studies of black yeasts have revealed a variety of reproductive strategies, from clonality to intense recombination and the formation of stable hybrids. Although a comprehensive understanding of the ecological role and molecular adaptations of halotolerant black yeasts remains elusive and the application of many experimental methods is challenging due to their slow growth and recalcitrant cell walls, much progress has been made in deciphering their halotolerance. Advances in molecular tools and genomics are once again accelerating the research of black yeasts, promising further insights into their survival strategies and the molecular basis of their adaptations. Key points • Black yeasts show remarkable adaptability to environmental stress • Black yeasts are part of microbial communities in hypersaline environments • Halotolerant black yeasts utilise various molecular and morphological adaptations

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Section: Genomics As the Future Of Black Yeast Researchmentioning

confidence: 99%

Black yeasts in hypersaline conditions

Gostinčar,

Gunde-Cimerman

2024

Appl Microbiol Biotechnol

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“…The current standard method for transforming A. pullulans is PEG-mediated protoplast transformation. Two research groups described CRISPR/Cas9-mediated genome editing in A. pullulans [19,20]. Zhang et al co-transformed two plasmids with yeast promoters and terminators: one for Cas9 expression and another for expressing a gRNA targeting the URA3 gene [20].…”

Section: Available Tools For a Pullulans Genome Editingmentioning

confidence: 99%

“…Kreuter et al used a ribonucleoprotein (RNP) approach, delivering the Cas9 enzyme and gRNAs in the cell during transformation [19]. They also successfully disrupted URA3 and improved homologous recombination efficiency [19].…”

Section: Available Tools For a Pullulans Genome Editingmentioning

confidence: 99%

“…We aimed for deletion of the gene coding for the orotidine-5-phosphate carboxylase (gene ID: 40743721) using a gRNA target described by Kreuter et al [19] (no. 14 in Supplementary Table 1).…”

Section: Construction Of the Crispr/cas9 Plasmid (Adapted From [28])mentioning

confidence: 99%

“…Two transformations were performed for which two different batches of protoplasts were used. The first transformation was made with frozen protoplasts only, and the second transformation was made with fresh and frozen protoplasts from the same batch [19].…”

Section: Protoplast Generation (Adapted From [19])mentioning

confidence: 99%

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Genomic deletions in Aureobasidium pullulans by an AMA1 plasmid for gRNA and CRISPR/Cas9 expression

Masi,

Wögerbauer,

Mach

et al. 2024

Fungal Biol Biotechnol

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Background Aureobasidium pullulans is a generalist polyextremotolerant black yeast fungus. It tolerates temperatures below 0 °C or salt concentrations up to 18%, among other stresses. A. pullulans genome sequencing revealed a high potential for producing bioactive metabolites. Only few molecular tools exist to edit the genome of A. pullulans, hence it is important to make full use of its potential. Two CRISPR/Cas9 methods have been proposed for the protoplast-based transformation of A. pullulans. These methods require the integration of a marker gene into the locus of the gene to be deleted, when the deletion of this gene does not yield a selectable phenotype. We present the adaptation of a plasmid-based CRISPR/Cas9 system developed in Aspergillus niger for A. pullulans to create deletion strains. Results The A. niger CRISPR/Cas9 plasmid led to efficient genomic deletions in A. pullulans. In this study, strains with deletions ranging from 30 to 862 bp were obtained by using an AMA1 plasmid-based genome editing strategy. Conclusion The CRISPR/Cas9 transformation system presented in this study provides new opportunities for strain engineering of A. pullulans. This system allows expression of Cas9 and antibiotic resistance while being easy to adapt. This strategy could open the path to intensive genomic engineering in A. pullulans.

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