Oligonucleotide-based CRISPR-Cas9 toolbox for efficient engineering of Komagataella phaffii.

Komagataella phaffii (Pichia pastoris) is a methylotrophic yeast that is favored by industry and academia mainly for expression of heterologous proteins. However, its full potential as a host for bioproduction of valuable compounds cannot be fully exploited as genetic tools are lagging behind those that are available for baker's yeast. The emergence of CRISPR-Cas9 technology has significantly improved the efficiency of gene manipulations of K.

A Mad7 System for Genetic Engineering of Filamentous Fungi.

The introduction of CRISPR technologies has revolutionized strain engineering in filamentous fungi. However, its use in commercial applications has been hampered by concerns over intellectual property (IP) ownership, and there is a need for implementing Cas nucleases that are not limited by complex IP constraints. One promising candidate in this context is the Mad7 enzyme, and we here present a versatile Mad7-CRISPR vector-set that can be efficiently used for the genetic engineering of four different Aspergillus species: Aspergillus nidulans, A.

A Versatile in Vivo DNA Assembly Toolbox for Fungal Strain Engineering.

Efficient homologous recombination in baker's yeast allows accurate fusion of DNA fragments via short identical sequence tags in vivo. Eliminating the need for an cloning step speeds up genetic engineering of this yeast and sets the stage for large high-throughput projects depending on DNA construction. With the aim of developing similar tools for filamentous fungi, we first set out to determine the genetic- and sequence-length requirements needed for efficient fusion reactions, and demonstrated that in nonhomologous end-joining deficient strains of , efficient fusions can be achieved by 25 bp sequence overlaps.

DIVERSIFY: A Fungal Multispecies Gene Expression Platform.

Recent sequencing of numerous fungal species revealed large repertoires of putative biotechnologically relevant genes and secondary metabolite gene clusters. However, often the commercial potential of these species is impeded by difficulties to predict host physiological and metabolic compatibility with a given product, and lack of adequate genetic tools. Consequently, most heterologous production is performed in standard hosts where genetic tools and experience are in place.

Cpf1 enables fast and efficient genome editing in Aspergilli.

Background: CRISPR technology has revolutionized fungal genetic engineering by increasing the speed and complexity of the experiments that can be performed. Moreover, the efficiency of the system often allows genetic engineering to be introduced in non-model species. The efficiency of CRISPR gene editing is due to the formation of specific DNA double-strand breaks made by RNA guided nucleases.

Efficient oligo nucleotide mediated CRISPR-Cas9 gene editing in Aspergilli.

CRISPR-Cas9 technologies are revolutionizing fungal gene editing. Here we show that survival of specific Cas9/sgRNA mediated DNA double strand breaks (DSBs) depends on the non-homologous end-joining, NHEJ, DNA repair pathway and we use this observation to develop a tool, TAPE, to assess protospacer efficiency in Aspergillus nidulans. Moreover, we show that in NHEJ deficient strains, highly efficient marker-free gene targeting can be performed.

CASCADE, a platform for controlled gene amplification for high, tunable and selection-free gene expression in yeast.

Over-expression of a gene by increasing its copy number is often desirable in the model yeast Saccharomyces cerevisiae. It may facilitate elucidation of enzyme functions, and in cell factory design it is used to increase production of proteins and metabolites. Current methods are typically exploiting expression from the multicopy 2 μ-derived plasmid or by targeting genes repeatedly into sequences like Ty or rDNA; in both cases, high gene expression levels are often reached.

Molecular and chemical characterization of the biosynthesis of the 6-MSA-derived meroterpenoid yanuthone D in Aspergillus niger.

Secondary metabolites in filamentous fungi constitute a rich source of bioactive molecules. We have deduced the genetic and biosynthetic pathway of the antibiotic yanuthone D from Aspergillus niger. Our analyses show that yanuthone D is a meroterpenoid derived from the polyketide 6-methylsalicylic acid (6-MSA).