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. niger, A. oryzae and A. campestris, the latter being a species that has never previously been genetically engineered. We successfully used Mad7 to introduce unspecific as well as specific template-directed mutations including gene disruptions, gene insertions and gene deletions. Moreover, we demonstrate that both single-stranded oligonucleotides and PCR fragments equipped with short and long targeting sequences can be used for efficient marker-free gene editing. Importantly, our CRISPR/Mad7 system was functional in both non-homologous end-joining (NHEJ) proficient and deficient strains. Therefore, the newly implemented CRISPR/Mad7 was efficient to promote gene deletions and integrations using different types of DNA repair in four different Aspergillus species, resulting in the expansion of CRISPR toolboxes in fungal cell factories.

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 Escherichia coli 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 Aspergillus nidulans, efficient fusions can be achieved by 25 bp sequence overlaps. Based on these results, we developed a novel fungal in vivo DNA assembly toolbox for simple and flexible genetic engineering of filamentous fungi. Specifically, we have used this method for construction of AMA1-based vectors, complex gene-targeting substrates for gene deletion and gene insertion, and for marker-free CRISPR based gene editing. All reactions were done via single-step transformations involving fusions of up to six different DNA fragments. Moreover, we show that it can be applied in four different species of Aspergilli. We therefore envision that in vivo DNA assembly can be advantageously used for many more purposes and will develop into a popular tool for fungal genetic engineering.

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. In filamentous fungi, only Cas9 has so far been used as the CRISPR nuclease. Since, gene editing with Cas9 is limited by its 5'-NGG-3' protospacer adjacent motif (PAM) sequence, it is important to introduce RNA guided nucleases that depend on other PAM sequences in order to be able to target a larger repertoire of genomic sites. Cpf1 from Lachnospiraceae bacterium employs a PAM sequence composed of 5'-TTTN-3' and therefore serves as an attractive option towards this goal. RESULTS: In this study we showed that Lb_cpf1 codon optimized for Aspergillus nidulans can be used for CRISPR based gene editing in filamentous fungi. We have developed a vector-based setup for Cpf1-mediated CRISPR experiments and showed that it works efficiently at different loci in A. nidulans and in A. niger. Specifically, we used our setup to demonstrate that Cpf1 is able to catalyze oligonucleotide-mediated genomic site-directed mutagenesis and marker-free gene targeting. CONCLUSIONS: In this paper we introduce Cpf1 as a new tool in the fungal CRISPR toolbox. Our experiments demonstrate that Cpf1 can be efficiently used in Aspergilli for gene editing thereby expanding the range of genomic DNA sequences that can be targeted by CRISPR technologies.

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. However, with 2 µ-based plasmid expression, the population of cells is very heterogeneous with respect to protein production; and for integration into repeated sequences it is difficult to determine the genetic setup of the resulting strains and to achieve specific gene doses. For both types of systems, the strains often suffer from genetic instability if proper selection pressure is not applied. Here we present a gene amplification system, CASCADE, which enables construction of strains with defined gene copy numbers. One or more genes can be amplified simultaneously and the resulting strains can be stably propagated on selection-free medium. As proof-of-concept, we have successfully used CASCADE to increase heterologous production of two fluorescent proteins, the enzyme ß-galactosidase the fungal polyketide 6-methyl salicylic acid and the plant metabolite vanillin glucoside.