Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus <i>Dichomitus squalens</i>
<i>Dichomitus squalens</i> 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 <i...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-10-01
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| Series: | Biomolecules |
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| Online Access: | https://www.mdpi.com/2218-273X/11/10/1526 |
| _version_ | 1797515210155622400 |
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| author | Joanna E. Kowalczyk Shreya Saha Miia R. Mäkelä |
| author_facet | Joanna E. Kowalczyk Shreya Saha Miia R. Mäkelä |
| author_sort | Joanna E. Kowalczyk |
| collection | DOAJ |
| description | <i>Dichomitus squalens</i> 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 <i>D. squalens</i> 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 <i>D. squalens</i>. 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 <i>D. squalens</i> and other wild-type (basidiomycete) fungi. |
| first_indexed | 2024-03-10T06:42:17Z |
| format | Article |
| id | doaj.art-44f649446d8d4cc6ba4d88f49324bb41 |
| institution | Directory Open Access Journal |
| issn | 2218-273X |
| language | English |
| last_indexed | 2024-03-10T06:42:17Z |
| publishDate | 2021-10-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomolecules |
| spelling | doaj.art-44f649446d8d4cc6ba4d88f49324bb412023-11-22T17:34:58ZengMDPI AGBiomolecules2218-273X2021-10-011110152610.3390/biom11101526Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus <i>Dichomitus squalens</i>Joanna E. Kowalczyk0Shreya Saha1Miia R. Mäkelä2Department of Microbiology, University of Helsinki, Viikinkaari 9, 00790 Helsinki, FinlandDepartment of Microbiology, University of Helsinki, Viikinkaari 9, 00790 Helsinki, FinlandDepartment of Microbiology, University of Helsinki, Viikinkaari 9, 00790 Helsinki, Finland<i>Dichomitus squalens</i> 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 <i>D. squalens</i> 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 <i>D. squalens</i>. 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 <i>D. squalens</i> and other wild-type (basidiomycete) fungi.https://www.mdpi.com/2218-273X/11/10/1526<i>Dichomitus squalens</i>CRISPR/Cas9genome editingribonucleoproteinsingle-stranded oligonucleotides |
| spellingShingle | Joanna E. Kowalczyk Shreya Saha Miia R. Mäkelä Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus <i>Dichomitus squalens</i> Biomolecules <i>Dichomitus squalens</i> CRISPR/Cas9 genome editing ribonucleoprotein single-stranded oligonucleotides |
| title | Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus <i>Dichomitus squalens</i> |
| title_full | Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus <i>Dichomitus squalens</i> |
| title_fullStr | Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus <i>Dichomitus squalens</i> |
| title_full_unstemmed | Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus <i>Dichomitus squalens</i> |
| title_short | Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus <i>Dichomitus squalens</i> |
| title_sort | application of crispr cas9 tools for genome editing in the white rot fungus i dichomitus squalens i |
| topic | <i>Dichomitus squalens</i> CRISPR/Cas9 genome editing ribonucleoprotein single-stranded oligonucleotides |
| url | https://www.mdpi.com/2218-273X/11/10/1526 |
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