Abstract
Ceriporiopsis subvermispora is a white-rot fungus with great potential for industrial and biotechnological applications, such as the pretreatment of lignocellulose in biorefineries, as it decomposes the lignin in the plant cell wall without causing severe cellulose degradation. A genetic transformation system was recently developed; however, gene-targeting experiments to disrupt or modify the gene(s) of interest remain challenging, and this is a bottleneck for further molecular genetic studies and breeding of C. subvermispora. Herein, we report efficient clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-assisted gene mutagenesis in this fungus. Two plasmids expressing Cas9 together with a different pyrG-targeting single-guide RNA were separately introduced into the monokaryotic C. subvermispora strain FP-90031-Sp/1, which frequently generated strains that exhibited resistance to 5-fluoroorotic acid and uridine/uracil auxotrophy. Southern blot analyses and genomic polymerase chain reaction followed by DNA sequencing of some mutants revealed that they were pyrG mutants. We also observed that hygromycin resistance of the pyrG mutants was frequently lost after repeated subcultivations, indicating that a maker-free genome editing occurred successfully. It is also suggested that a gene mutation(s) can be introduced via a transient expression of Cas9 and a single-guide RNA; this feature, together with high-frequency gene targeting using the CRISPR/Cas9 system, would be helpful for studies on lignocellulose-degrading systems in C. subvermispora.
Key points
• Efficient plasmid-based CRISPR/Cas9 was established in C. subvermispora.
• The mutations can be introduced via a transient expression of Cas9 and sgRNA.
• A maker-free CRISPR/Cas9 is established in this fungus.
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Data availability
All data supporting the claims of this manuscript are presented and made available in this manuscript and Supplemental Table S1.
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Acknowledgements
We would like to thank Prof. Keishi Osakabe (Tokushima University, Japan) for providing plasmid pCcPef3-126.
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This work was supported in part by the Grants-in-Aid for Scientific Research (KAKENHI 18H02254, 18KK0178, 19K22332, and 21K18224 to Y.H.: 19F19095 to Y.H. and H.X.: 20F20092 to Y.H. and D.X.N.), and JSPS Bilateral Program (JPJSBP 120208402 and 120209920 to Y.H.).
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T.N. and Y.H. conceived and designed the study. T.N., C.I., and D.X.N conducted the experiments. T.N. and C.I. performed the analyses. T.N., M.K., M.S., and Y.H. drafted the manuscript. The present work has never been achieved without efforts of all the coauthors. All authors read and approved the final manuscript.
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Nakazawa, T., Inoue, C., Nguyen, D.X. et al. CRISPR/Cas9 using a transient transformation system in Ceriporiopsis subvermispora. Appl Microbiol Biotechnol 106, 5575–5585 (2022). https://doi.org/10.1007/s00253-022-12095-7
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DOI: https://doi.org/10.1007/s00253-022-12095-7