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Abstract
Dichomitus squalens is an emerging reference species that can be used to investigate white-rot fungal plant biomass degradation, as it has flexible physiology to utilize different types of biomass as sources of carbon and energy. Recent comparative (post-) genomic studies on D. squalens resulted in an increasingly detailed knowledge of the genes and enzymes involved in the lignocellulose breakdown in this fungus and showed a complex transcriptional response in the presence of lignocellulose-derived compounds. To fully utilize this increasing amount of data, efficient and reliable genetic manipulation tools are needed, e.g., to characterize the function of certain proteins in vivo and facilitate the construction of strains with enhanced lignocellulolytic capabilities. However, precise genome alterations are often very difficult in wild-type basidiomycetes partially due to extremely low frequencies of homology directed recombination (HDR) and limited availability of selectable markers. To overcome these obstacles, we assessed various Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) -based strategies for selectable homology and non-homologous end joining (NHEJ) -based gene editing in D. squalens. We also showed an induction of HDR-based genetic modifications by using single-stranded oligodeoxynucleotides (ssODNs) in a basidiomycete fungus for the first time. This paper provides directions for the application of targeted CRISPR/Cas9-based genome editing in D. squalens and other wild-type (basidiomycete) fungi.
Highlights
Plant biomass degrading filamentous fungi are essential nutrient cyclers in terrestrial environments and important producers of enzymes and metabolites, for example, in different industrial sectors [1]
In this work we evaluated different methods for CRISPR/Cas9-based gene editing in the wild-type D. squalens strain (WT) D. squalens (Figure 1)
We chose to target this gene with the CRISPR/Cas9 system in WT D. squalens
Summary
Plant biomass degrading filamentous fungi are essential nutrient cyclers in terrestrial environments and important producers of enzymes and metabolites, for example, in different industrial sectors [1] Due to their ecological and societal relevance, the number of whole genome-sequenced fungal species and strains is exponentially increasing together with post-genomic studies [2]. The discovery of CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease form Streptococcus pyogenes) and its application to genome engineering [4], have revolutionized research on several scientific areas in less than a decade The success of this technology is based on the ability of the Cas nuclease to recognize and cut a specific DNA sequence in the genome.
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