Aspergillus nidulans, also called Emericella nidulans, is one of the critical fungal systems in genetics and cell biology. It has been an important research organism for studying eukaryotic cell biology and a wide range of subjects including recombination,DNA repair,mutation, cellcycle control, tubulin, chromatin, nucleokinesis, pathogenesis, metabolism, and experimental evolution.Fungi play a major role in recirculating biomass in ecosystems, as they degrade all types of organic matter. For this reason, they serve as a major source of industrially relevant enzymes, e.g. amylases, cellulases, lipases, pectinases, and proteases. The number of fully sequenced fungal genomes is rapidly increasing. Since genetic tools are poorly developed for most filamentous fungi, it is currently difficult to employ genetic engineering for understanding the biology of these fungi and to fully exploit them industrially. Therefore, to developing versatile methods that can be used to genetically manipulate non-model filamentous fungi, scientists have developed a CRISPR-Cas9 based system adapted for use in filamentous fungi. 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. Aspergillus nidulans (Emericella nidulans) have a long and productive history as a source of industrial chemicals and enzymes and as a developmental model system to study genetic regulation, developmental biology, signal transduction, and secondary metabolism. So it will be a new pathway to study Aspergillus nidulans by conducting gene knockouts, gene knock-in or point mutations.
Applying CRISPR in highly-efficient marker-free gene targeting in Aspergillus nidulans
The 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, in NHEJ deficient strains, highly efficient marker-free gene targeting can be performed. Indeed, it was showed in the study that even single-stranded oligonucleotides efficiently work as repair templates of specific Cas9/sgRNA induced DNA DSBs in A. nidulans, A. niger, indicating that this type of repair may be wide-spread in filamentous fungi. Importantly, by using single-stranded oligonucleotides for CRISPR-Cas9 mediated gene editing, it is possible to introduce specific point mutations as well gene deletions (gene knockout) at efficiencies approaching 100%. Therefore, it is possible to introduce two-point mutations and one-gene insertion in one transformation experiment with very high efficiency.
Cpf1 enables fast and efficient genome editing in Aspergilli
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 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. In this study, the Lb_cpf1 codon-optimized for Aspergillus nidulans can be used for CRISPR based gene editing in filamentous fungi. Researchers developed a vector-based setup for Cpf1-mediated CRISPR experiments and showed that it works efficiently at different loci in A. nidulans and A. niger. Specifically, the Cpf1 can catalyze oligonucleotide-mediated genomic site-directed mutagenesis and marker-free gene targeting. The results showed 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.
Efficient CRISPR knockout in A. nidulans
Gene targeting by homologous recombination during transformation is possible in A. nidulans, but the frequency of correct gene targeting is variable and often low. Researchers identified the A. nidulans homolog (nkuA) of the human KU70 gene that is essential for nonhomologous end-joining of DNA in double-strand break repair. Deletion of nkuA (nkuAΔ) greatly reduces the frequency of nonhomologous integration of transforming DNA fragments, leading to dramatically improved gene targeting. The selectable heterologous markers were also developed in A. nidulans but do not direct integration at any site in the A. nidulans genome. In combination, nkuAΔ and the heterologous selectable markers make up a very efficient gene-targeting system. In experiments involving scores of genes, 90% or more of the transformants carried a single insertion of the transforming DNA at the correct site. The system works with linear and circular transforming molecules and it works for tagging genes with fluorescent moieties, replacing genes, and replacing promoters. This system is efficient enough to make genome-wide gene-targeting projects in A. nidulans feasible.
Ubigene developed CRISPR-B™ which optimizes the microbial gene-editing vectors and process. The efficiency and accuracy are much higher than traditional methods. CRISPR-B™ can be used in gene editing of bacteria and fungi. Easily achieve microbial gene knockout (KO), point mutation (PM), and knockin (KI).
1. Nødvig CS, Hoof JB, Kogle ME, et al. Efficient oligonucleotide mediated CRISPR-Cas9 gene editing in Aspergilli. Fungal Genet Biol. 2018;115:78-89.
2. Nødvig CS, Nielsen JB, Kogle ME, Mortensen, UH. A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi. PLoS One. 2015;10(7):e0133085. Published 2015 Jul 15.
3. Vanegas, K.G., Jarczynska, Z.D., Strucko, T. et al. Cpf1 enables fast and efficient genome editing in Aspergilli. Fungal Biol Biotechnol 6, 6 (2019).
4. Nayak T, Szewczyk E, Oakley CE, et al. A versatile and efficient gene-targeting system for Aspergillus nidulans. Genetics. 2006;172(3):1557-1566.
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