Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus Dichomitus squalens

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.

The lignin‐degrading abilities of Gelatoporia subvermispora gat1 and pex1 mutants generated via CRISPR/Cas9

“…Contrary to homologous recombination‐based gene targeting, CRISPR/Cas9‐based gene targeting would be a realistic molecular genetic approach for various white‐rot fungi because it is applicable as long as genetic transformation is available. As CRISPR/Cas9 can now be used on some other white‐rot fungi, such as Ganoderma lucidum and Dichomitus squalens (Kowalczyk et al, 2021; Qin et al, 2017), current difficulties in performing molecular genetic studies of these species may be resolved in the near future. Such a development would promote the use of lignocellulose degradation by the white‐rot fungus as an important step toward the eco‐friendly processing of lignocellulosic biomass.…”

Systems biology-guided understanding of white-rot fungi for biotechnological applications: A review

“…Nonetheless, a genetic toolkit for WRF is essential to validate systems biology observations, gene-function relationships, and further improve their performance. Progress on the development of efficient genetic tools (i.e., CRISPR/Cas9) has been recently published for few species of WRF such as P. ostreatus ( Boontawon et al., 2021 ), G. lucidum ( Wang et al., 2020 ), and D. squalens ( Kowalczyk et al., 2021 ), which is a significant advance in fungal biology and proves the suitability of WRF for further metabolic engineering efforts.…”

Pleurotus ostreatus as a model mushroom in genetics, cell biology, and material sciences

“…The RNP-based method has been reported for some agaricomycetes, such as S. commune (Jan Vonk et al 2019 ), P. ostreatus (Boontawon et al 2021b ), Dichomitus squalens (Kowalczyk et al 2021 ), F. filiformis (Liu et al 2022b ), C. cinerea (Pareek et al 2022 ), and G. lucidum (Eom et al 2023 ) (Table 1 ). Technically, this protocol can be applied to species lacking a suitable selection marker gene, provided that protoplasts can be generated efficiently.…”

“…However, at present, the efficiency of genome editing using RNP-based methods may be lower than that of DNA-based methods (Boontawon et al 2021b ). The major challenge of the RNP-based method is the lack of an efficient selection system; this method has been applied only to a special target gene whose disruption makes it possible to screen for, such as pyrG (Boontawon et al 2021b ; Kowalczyk et al 2021 ; Eom et al 2023 ) or fcy1 (Boontawon et al 2023 ), unless a foreign selection marker was also introduced (Jan Vonk et al 2019 ; Boontawon et al 2021b ; Kowalczyk et al 2021 ; Pareek et al 2022 ). To overcome this problem, RNP-dependent CRISPR/Cas9 with two target genes, one for screening and the other the gene of interest, was developed in P. ostreatus (Boontawon et al 2023 ).…”