A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi

Abstract

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. For that reason there is a demand for developing versatile methods that can be used to genetically manipulate non-model filamentous fungi. To facilitate this, we have developed a CRISPR-Cas9 based system adapted for use in filamentous fungi. The system is simple and versatile, as RNA guided mutagenesis can be achieved by transforming a target fungus with a single plasmid. The system currently contains four CRISPR-Cas9 vectors, which are equipped with commonly used fungal markers allowing for selection in a broad range of fungi. Moreover, we have developed a script that allows identification of protospacers that target gene homologs in multiple species to facilitate introduction of common mutations in different filamentous fungi. With these tools we have performed RNA-guided mutagenesis in six species of which one has not previously been genetically engineered. Moreover, for a wild-type Aspergillus aculeatus strain, we have used our CRISPR Cas9 system to generate a strain that contains an AACU_pyrG marker and demonstrated that the resulting strain can be used for iterative gene targeting.

Tet-on, or Tet-off, that is the question: Advanced conditional gene expression in Aspergillus

“…Such synthetic biological approaches include reengineering a prokaryotic serine recombinase for site-specific marker recycling (Hartmann et al, 2010), the CRISPR-Cas9 system for genome editing (Vyas et al, 2015) ( Nødvig et al, 2015), generation of RNA interference vectors for gene knock-down (Skowyra and Doering, 2012) and an impressive range of inducible promoter systems from bacteria and other kingdoms (Hörner and Weber, 2012). Such conditional expression systems, where transcript abundance is controlled by experimental parameters, are a vital tool for characterisation of essential genes, which are unable to be analysed by deletion strategies.…”

Multiplex gene editing of the Yarrowia lipolytica genome using the CRISPR-Cas9 system

“…The type II CRISPR-Cas9 system has been extensively developed as a powerful genome-editing tool because of its high on-target efficiency in numerous prokaryotes and eukaryotes, including (but not limited to) E. coli [16], Streptomyces spp. [7], Clostridium cellulolyticum [43], Lactobacillus reuteri [30], Saccharomyces cerevisiae [11], Bombyx mori [41], Filamentous fungi [23,28], Drosophila [46], Candida albicans [40], higher plants [32], and multiple human cell lines [8,19,25]. The CRISPR-associated protein (Cas9) endonuclease is guided by a mature CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) towards a target DNA sequence (known as a protospacer).…”

Development of the CRISPR/Cas9 System for Targeted Gene Disruption in Aspergillus fumigatus

“…Remarkably, and in just the past 2 years, the system derived from Streptococcus pyogenes (17) has been simplified as a genomic engineering tool in diverse eukaryotes, such as mice and rats (18)(19)(20)(21), plants (22,23), and even fungi, including Saccharomyces cerevisiae (24), Candida albicans (25), Trichoderma reesei (26), and other Aspergillus spp. (27). Briefly, a synthetic singleguide RNA (gRNA), which is a fusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), associates with endonuclease Cas9 and guides it to a 20-nucleotide genomic target.…”