{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,11]],"date-time":"2026-08-11T09:35:35Z","timestamp":1786440935871,"version":"build-2736575974"},"reference-count":125,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,8,11]],"date-time":"2026-08-11T00:00:00Z","timestamp":1786406400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"FGV Agri Services","award":["FR-0604-80-0219"],"award-info":[{"award-number":["FR-0604-80-0219"]}]},{"name":"FGV Agri Services","award":["FR-0604-80-0225"],"award-info":[{"award-number":["FR-0604-80-0225"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["J. Fungi","JoF"],"abstract":"<jats:p>Oil palm is a major commodity crop in Southeast Asia, particularly in Malaysia and Indonesia, but its productivity is severely threatened by basal stem rot (BSR) and upper stem rot (USR) caused by the white-rot fungus Ganoderma boninense. Infected palms can lose up to 80% yield and die within 6\u201324 months (young) or 2\u20133 years (mature). Despite extensive field management efforts, disease incidence continues to rise, especially after replanting. Understanding infection mechanisms and validating fungal virulence factors are crucial for effective control, yet functional genomics in G. boninense has been limited. Previous RNAi-based gene silencing provided initial insights but was constrained by off-target effects and transient activity. Here, we report the first successful application of CRISPR\/Cas9 genome editing in G. boninense for functional gene studies. Two genes were targeted: pyrG, essential in uridine monophosphate (UMP) biosynthesis; and hyd-2, encoding a hydrophobin, a potential virulence determinant implicated in host invasion. The disruption of pyrG produced a uracil auxotroph, and the knockout was validated by screening on 5-FOA and validated our system as a functional molecular tool. Disruption of hyd-2 reduced infection capability of the fungus by ~72% to ~91% in vitro. Mutations in both gene disruptions, including insertions, deletions, and substitutions, were confirmed by sequencing. Sequencing analysis also revealed incomplete editing events, as wild-type gene sequences were detected alongside edited alleles in the mutants. Future enhancements should focus on improving editing efficiency of the system. This work establishes a robust platform for functional genetic analysis and dissecting pathogenicity in G. boninense, ultimately advancing strategies to mitigate basal stem rot disease in oil palm.<\/jats:p>","DOI":"10.3390\/jof12080594","type":"journal-article","created":{"date-parts":[[2026,8,11]],"date-time":"2026-08-11T08:09:36Z","timestamp":1786435776000},"page":"594","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Establishment of CRISPR\/Cas9 Genome Editing in Ganoderma boninense and Functional Validation of Hydrophobin-2 as a Virulence Determinant"],"prefix":"10.3390","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-9629-1193","authenticated-orcid":false,"given":"Anis Farhan Fatimi","family":"Ab Wahab","sequence":"first","affiliation":[{"name":"Geoinformatics, Genomics & Bioinformatics Department, FGV Innovation Centre (Biotechnology), FGV R&D Sdn. Bhd., PT. 23417 Lengkuk Teknologi, Bandar Enstek 71760, Negeri Sembilan, Malaysia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6104-2739","authenticated-orcid":false,"given":"Mohd Azinuddin Ahmad","family":"Mokhtar","sequence":"additional","affiliation":[{"name":"Plant Breeding Unit, Tun Razak Agricultural Research Centre (PPPTR), FGV R&D Sdn. Bhd., Jerantut 27000, Pahang, Malaysia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7833-0622","authenticated-orcid":false,"given":"Sharmilah","family":"Vetaryan","sequence":"additional","affiliation":[{"name":"Geoinformatics, Genomics & Bioinformatics Department, FGV Innovation Centre (Biotechnology), FGV R&D Sdn. Bhd., PT. 23417 Lengkuk Teknologi, Bandar Enstek 71760, Negeri Sembilan, Malaysia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2750-6701","authenticated-orcid":false,"given":"Yang Ping","family":"Lee","sequence":"additional","affiliation":[{"name":"Geoinformatics, Genomics & Bioinformatics Department, FGV Innovation Centre (Biotechnology), FGV R&D Sdn. Bhd., PT. 23417 Lengkuk Teknologi, Bandar Enstek 71760, Negeri Sembilan, Malaysia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Midot, F., Lau, S.Y.L., Wong, W.C., Tung, H.J., Yap, M.L., Lo, M.L., Jee, M.S., Dom, S.P., and Melling, L. (2019). Genetic diversity and demographic history of Ganoderma boninense in oil palm plantations of Sarawak, Malaysia inferred from ITS regions. Microorganisms, 7.","DOI":"10.3390\/microorganisms7100464"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Hussain, H., Aspar, H.M., Mazlan, R., Nadras, S., Ab Rahman, M.A.K., Hassan, N.A.M., and Minal, J. (2025). Palm Oil Business: Global Market Trend and Local Outlook. The Palm Oil Export Market, Routledge.","DOI":"10.4324\/9781003518600-2"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Jazuli, N.A., Kamu, A., Chong, K.P., Gabda, D., Hassan, A., Abu Seman, I., and Ho, C.M. (2022). A review of factors affecting Ganoderma basal stem rot disease progress in oil palm. Plants, 11.","DOI":"10.3390\/plants11192462"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"103671","DOI":"10.1016\/j.envsci.2024.103671","article-title":"Palm oil expansion in Malaysia and its countermeasures through policy window and biorefinery approach","volume":"153","author":"Hassan","year":"2024","journal-title":"Environ. Sci. Policy"},{"key":"ref_5","first-page":"159","article-title":"Oil palm economic performance in Malaysia and R&D progress in 2019","volume":"32","author":"Parveez","year":"2020","journal-title":"J. Oil Palm. Res."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Naidu, L., and Moorthy, R. (2021). A review of key sustainability issues in Malaysian palm oil industry. Sustainability, 13.","DOI":"10.3390\/su131910839"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Gesteiro, E., Guijarro, L., S\u00e1nchez-Muniz, F.J., Vidal-Carou, M.d.C., Troncoso, A., Venanci, L., Jimeno, V., Quilez, J., Anad\u00f3n, A., and Gonz\u00e1lez-Gross, M. (2019). Palm oil on the edge. Nutrients, 11.","DOI":"10.3390\/nu11092008"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"109","DOI":"10.18280\/ijsdp.170110","article-title":"Sustainability of the Palm Oil Industry: An Empirical Study of the Development of Sustainable Oil Palm in Bengkalis Regency, Indonesia","volume":"17","author":"Hariyanti","year":"2022","journal-title":"Int. J. Sustain. Dev. Plan"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1007\/s42398-022-00244-7","article-title":"Ganoderma boninense basal stem rot induced by climate change and its effect on oil palm","volume":"5","author":"Abubakar","year":"2022","journal-title":"Environ. Sustain."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"603","DOI":"10.1094\/PDIS-02-22-0358-FE","article-title":"Basal stem rot of oil palm: The pathogen, disease incidence, and control methods","volume":"107","author":"Zakaria","year":"2023","journal-title":"Plant Dis."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"10316","DOI":"10.1038\/s41598-023-37507-0","article-title":"Exploring the potential of Bornean polypore fungi as biological control agents against pathogenic Ganoderma boninense causing basal stem rot in oil palm","volume":"13","author":"Darlis","year":"2023","journal-title":"Sci. Rep."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Khoo, Y.W., and Chong, K.P. (2023). Ganoderma boninense: General characteristics of pathogenicity and methods of control. Front. Plant Sci., 14.","DOI":"10.3389\/fpls.2023.1156869"},{"key":"ref_13","unstructured":"Virdiana, I., Hasan, Y., Aditya, R., and Flood, J. (2010). Testing the effects of oil palm replanting practices (windrowing, fallowing and poisoning) on incidence of Ganoderma. Proc. IOPC, 7."},{"key":"ref_14","first-page":"517","article-title":"Early detection and management of Ganoderma basal stem rot disease: A special report from sabah","volume":"3","author":"Chong","year":"2016","journal-title":"Trans. Sci. Technol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1007\/s11046-004-4438-0","article-title":"Enhancing biological control of basal stem rot disease (Ganoderma boninense) in oil palm plantations","volume":"159","author":"Susanto","year":"2005","journal-title":"Mycopathologia"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"43","DOI":"10.55230\/mabjournal.v51i5.2347","article-title":"Evaluation and improvement of protocols for Ganoderma boninense protoplast isolation and regeneration","volume":"51","author":"Zairun","year":"2022","journal-title":"Malays. Appl. Biol."},{"key":"ref_17","first-page":"12","article-title":"Selection for partial resistance in oil palm progenies to Ganoderma basal stem rot","volume":"16","author":"Idris","year":"2004","journal-title":"J. Oil Palm. Res."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"982","DOI":"10.1111\/j.1365-3059.2009.02100.x","article-title":"Basal stem rot of oil palm (Elaeis guineensis); mode of root infection and lower stem invasion by Ganoderma boninense","volume":"58","author":"Rees","year":"2009","journal-title":"Plant Pathol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"567","DOI":"10.1111\/j.1365-3059.2011.02533.x","article-title":"Ganoderma boninense basidiospores in oil palm plantations: Evaluation of their possible role in stem rots of Elaeis guineensis","volume":"61","author":"Rees","year":"2012","journal-title":"Plant Pathol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"87","DOI":"10.62872\/rdjwg922","article-title":"Environmental sustainability and economic growth: Policy implications for developing countries","volume":"1","author":"Utomo","year":"2024","journal-title":"Nomico J."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Dhillon, B., Hamelin, R.C., and Rollins, J.A. (2021). Transcriptional profile of oil palm pathogen, Ganoderma boninense, reveals activation of lignin degradation machinery and possible evasion of host immune response. BMC Genom., 22.","DOI":"10.1186\/s12864-021-07644-9"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Khairi, M.H.F., Nor Muhammad, N.A., Bunawan, H., Abdul Murad, A.M., and Ramzi, A.B. (2022). Unveiling the core effector proteins of oil palm pathogen Ganoderma boninense via pan-secretome analysis. J. Fungi, 8.","DOI":"10.20944\/preprints202206.0376.v1"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"113198","DOI":"10.1016\/j.scienta.2024.113198","article-title":"Gene silencing of Ganoderma boninense Lanosterol 14\u03b1-demethylase (GBERG11) affects ergosterol biosynthesis and pathogenicity towards oil palm","volume":"332","author":"Lim","year":"2024","journal-title":"Sci. Hortic."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1016\/j.fbr.2016.05.003","article-title":"RNAi silencing: A tool for functional genomics research on fungi","volume":"30","year":"2016","journal-title":"Fungal Biol. Rev."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"656","DOI":"10.1016\/j.tplants.2011.08.006","article-title":"New dimensions for VIGS in plant functional genomics","volume":"16","author":"Mysore","year":"2011","journal-title":"Trends Plant Sci."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Svoboda, P. (2020). Key mechanistic principles and considerations concerning RNA interference. Front. Plant Sci., 11.","DOI":"10.3389\/fpls.2020.01237"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"7079","DOI":"10.1007\/s11033-022-07442-w","article-title":"CRISPR\/Cas9 a simple, inexpensive and effective technique for gene editing","volume":"49","author":"Ferreira","year":"2022","journal-title":"Mol. Biol. Rep."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Janik, E., Niemcewicz, M., Ceremuga, M., Krzowski, L., Saluk-Bijak, J., and Bijak, M. (2020). Various aspects of a gene editing system\u2014Crispr\u2013cas9. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21249604"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"68","DOI":"10.1186\/s43141-020-00078-y","article-title":"The genome editing revolution","volume":"18","author":"Khalil","year":"2020","journal-title":"J. Genet. Eng. Biotechnol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"105338","DOI":"10.1016\/j.jddst.2024.105338","article-title":"Principles of CRISPR-Cas9 technology: Advancements in genome editing and emerging trends in drug delivery","volume":"92","author":"Aljabali","year":"2024","journal-title":"J. Drug Deliv. Sci. Technol."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Khan, M.S., Qureshi, N., Khan, R., Son, Y.-O., and Maqbool, T. (2025). CRISPR\/Cas9-Based therapeutics as a promising strategy for management of Alzheimer\u2019s disease: Progress and prospects. Front. Cell. Neurosci., 19.","DOI":"10.3389\/fncel.2025.1578138"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1061","DOI":"10.36347\/sajb.2025.v13i08.002","article-title":"CRISPR Applications in Enhancing Medicinal Plant Yields","volume":"8","author":"Afridi","year":"2025","journal-title":"Sch. Acad. J. Biosci."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1016\/j.fgb.2015.09.001","article-title":"Genome editing in Ustilago maydis using the CRISPR\u2013Cas system","volume":"89","author":"Schuster","year":"2016","journal-title":"Fungal Genet. Biol."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"\u00d6kmen, B., Schwammbach, D., Bakkeren, G., Neumann, U., and Doehlemann, G. (2021). The Ustilago hordei\u2013barley interaction is a versatile system for characterization of fungal effectors. J. Fungi, 7.","DOI":"10.3390\/jof7020086"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Lu, S., Wang, Y., Shen, X., Guo, F., Zhou, C., Li, R., and Chen, B. (2021). SsPEP1, an effector with essential cellular functions in sugarcane smut fungus. J. Fungi, 7.","DOI":"10.3390\/jof7110954"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"386","DOI":"10.1111\/1751-7915.13534","article-title":"Dual sgRNA-directed gene deletion in basidiomycete Ganoderma lucidum using the CRISPR\/Cas9 system","volume":"13","author":"Liu","year":"2020","journal-title":"Microb. Biotechnol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"e01510-21","DOI":"10.1128\/AEM.01510-21","article-title":"Targeted gene insertion and replacement in the basidiomycete Ganoderma lucidum by inactivation of nonhomologous end joining using CRISPR\/Cas9","volume":"87","author":"Tu","year":"2021","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"689","DOI":"10.1016\/j.procbio.2022.08.015","article-title":"CRISPR-Cas9 assisted in-situ complementation of functional genes in the basidiomycete Ganoderma lucidum","volume":"121","author":"Wang","year":"2022","journal-title":"Process Biochem."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"e2409006","DOI":"10.71150\/jm.2409006","article-title":"Simultaneous gene editing of both nuclei in a dikaryotic strain of Ganoderma lucidum using Cas9-gRNA ribonucleoprotein","volume":"63","author":"Choi","year":"2025","journal-title":"J. Microbiol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"164","DOI":"10.1016\/0076-6879(87)54076-9","article-title":"[10] 5-Fluoroorotic acid as a selective agent in yeast molecular genetics","volume":"Volume 154","author":"Boeke","year":"1987","journal-title":"Methods in Enzymology"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"603","DOI":"10.1007\/s42770-019-00083-5","article-title":"Hydrophobin HFBII-4 from Trichoderma asperellum induces antifungal resistance in poplar","volume":"50","author":"Zhang","year":"2019","journal-title":"Braz. J. Microbiol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"2610","DOI":"10.1038\/s41396-020-0709-0","article-title":"Evolutionary compromises in fungal fitness: Hydrophobins can hinder the adverse dispersal of conidiospores and challenge their survival","volume":"14","author":"Cai","year":"2020","journal-title":"ISME J."},{"key":"ref_43","first-page":"79","article-title":"Hydrophobins, the fungal coat unravelled","volume":"1469","year":"2000","journal-title":"Biochim. Biophys. Acta BBA-Rev. Biomembr."},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Blango, M.G., Kniemeyer, O., and Brakhage, A.A. (2019). Conidial surface proteins at the interface of fungal infections. PLoS Pathog., 15.","DOI":"10.1371\/journal.ppat.1007939"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"fnad042","DOI":"10.1093\/femsle\/fnad042","article-title":"Development of a gene-targeting system using CRISPR\/Cas9 and utilization of pyrG as a novel selectable marker in Lentinula edodes","volume":"370","author":"Kamiya","year":"2023","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1016\/j.jgg.2015.04.005","article-title":"Orotidine Monophosphate Decarboxylase\u2013A Fascinating Workhorse Enzyme with Therapeutic Potential","volume":"42","author":"Fujihashi","year":"2015","journal-title":"J. Genet. Genom."},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Rojas-Osnaya, J., Quintana-Quirino, M., Espinosa-Valencia, A., Bravo, A.L., and N\u00e1jera, H. (2024). Hydrophobins: Multitask proteins. Front. Phys., 12.","DOI":"10.3389\/fphy.2024.1393340"},{"key":"ref_48","doi-asserted-by":"crossref","unstructured":"Carter-House, D., Stajich, J., Unruh, S., and Kurbessoian, T. (2020). Fungal CTAB DNA Extraction, Springer.","DOI":"10.17504\/protocols.io.bhx8j7rw"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"012032","DOI":"10.1088\/1755-1315\/948\/1\/012032","article-title":"An optimized method for high-quality DNA extraction medicinal fungi Mycoleptodonoides aitchisonii for whole genome sequencing","volume":"948","author":"Riffiani","year":"2021","journal-title":"Proc. IOP Conf. Ser. Earth Environ. Sci."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"e58","DOI":"10.1093\/nar\/gky164","article-title":"Easy quantification of template-directed CRISPR\/Cas9 editing","volume":"46","author":"Brinkman","year":"2018","journal-title":"Nucleic Acids Res."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1089\/crispr.2021.0113","article-title":"Inference of CRISPR edits from Sanger trace data","volume":"5","author":"Conant","year":"2022","journal-title":"Cris. J."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"1023","DOI":"10.1038\/s41587-021-01156-3","article-title":"SignalP 6.0 predicts all five types of signal peptides using protein language models","volume":"40","author":"Teufel","year":"2022","journal-title":"Nat. Biotechnol."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"1690","DOI":"10.1093\/bioinformatics\/btx818","article-title":"DeepSig: Deep learning improves signal peptide detection in proteins","volume":"34","author":"Savojardo","year":"2018","journal-title":"Bioinformatics"},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"W429","DOI":"10.1093\/nar\/gkm256","article-title":"Advantages of combined transmembrane topology and signal peptide prediction\u2014The Phobius web server","volume":"35","author":"Krogh","year":"2007","journal-title":"Nucleic Acids Res."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1093\/sysbio\/sys029","article-title":"MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space","volume":"61","author":"Ronquist","year":"2012","journal-title":"Syst. Biol."},{"key":"ref_56","doi-asserted-by":"crossref","unstructured":"Quarantin, A., Hadeler, B., Kr\u00f6ger, C., Sch\u00e4fer, W., Favaron, F., Sella, L., and Mart\u00ednez-Rocha, A.L. (2019). Different hydrophobins of Fusarium graminearum are involved in hyphal growth, attachment, water-air interface penetration and plant infection. Front. Microbiol., 10.","DOI":"10.3389\/fmicb.2019.00751"},{"key":"ref_57","doi-asserted-by":"crossref","unstructured":"Bayry, J., Aimanianda, V., Guijarro, J.I., Sunde, M., and Latge, J.-P. (2012). Hydrophobins\u2014Unique fungal proteins. PLoS Pathog., 8.","DOI":"10.1371\/journal.ppat.1002700"},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"fnad051","DOI":"10.1093\/femsle\/fnad051","article-title":"Function of a hydrophobin in growth and development, nitrogen regulation, and abiotic stress resistance of Ganoderma lucidum","volume":"370","author":"Qiao","year":"2023","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"2581","DOI":"10.4049\/jimmunol.1300748","article-title":"The rodA hydrophobin on Aspergillus fumigatus spores masks dectin-1\u2013and dectin-2\u2013dependent responses and enhances fungal survival in vivo","volume":"191","author":"Carrion","year":"2013","journal-title":"J. Immunol."},{"key":"ref_60","doi-asserted-by":"crossref","unstructured":"Valsecchi, I., Dupres, V., Stephen-Victor, E., Guijarro, J., Gibbons, J., Beau, R., Bayry, J., Coppee, J., Lafont, F., and Latg\u00e9, J. (2017). Role of hydrophobins in Aspergillus fumigatus. J. Fungi, 4.","DOI":"10.3390\/jof4010002"},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"e02442-22","DOI":"10.1128\/mbio.02442-22","article-title":"Comparative transcriptomics of Fusarium graminearum and Magnaporthe oryzae spore germination leading up to infection","volume":"14","author":"Cavinder","year":"2023","journal-title":"mBio"},{"key":"ref_62","doi-asserted-by":"crossref","unstructured":"Tanaka, T., Terauchi, Y., Yoshimi, A., and Abe, K. (2022). Aspergillus hydrophobins: Physicochemical properties, biochemical properties, and functions in solid polymer degradation. Microorganisms, 10.","DOI":"10.3390\/microorganisms10081498"},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"464","DOI":"10.1089\/hum.2016.009","article-title":"CRISPRs for optimal targeting: Delivery of CRISPR components as DNA, RNA, and protein into cultured cells and single-cell embryos","volume":"27","author":"Kouranova","year":"2016","journal-title":"Hum. Gene Ther."},{"key":"ref_64","doi-asserted-by":"crossref","unstructured":"Saikia, B., Remya, S., Debbarma, J., Maharana, J., Sastry, G.N., and Chikkaputtaiah, C. (2024). CRISPR\/Cas9-based genome editing and functional analysis of SlHyPRP1 and SlDEA1 genes of Solanum lycopersicum L. in imparting genetic tolerance to multiple stress factors. Front. Plant Sci., 15.","DOI":"10.3389\/fpls.2024.1304381"},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"841","DOI":"10.4161\/rna.24203","article-title":"crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus","volume":"10","author":"Karvelis","year":"2013","journal-title":"RNA Biol."},{"key":"ref_66","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1146\/annurev-genet-071719-022559","article-title":"The tracrRNA in CRISPR biology and technologies","volume":"55","author":"Liao","year":"2021","journal-title":"Annu. Rev. Genet."},{"key":"ref_67","doi-asserted-by":"crossref","first-page":"489","DOI":"10.1038\/s41467-022-28137-7","article-title":"Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage","volume":"13","author":"Riesenberg","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_68","doi-asserted-by":"crossref","unstructured":"Li, J., Kong, D., Ke, Y., Zeng, W., and Miki, D. (2024). Application of multiple sgRNAs boosts efficiency of CRISPR\/Cas9-mediated gene targeting in Arabidopsis. BMC Biol., 22.","DOI":"10.1186\/s12915-024-01810-7"},{"key":"ref_69","doi-asserted-by":"crossref","first-page":"7489","DOI":"10.1038\/s41598-017-07871-9","article-title":"CRISPR\/Cas9-mediated genome editing efficiently creates specific mutations at multiple loci using one sgRNA in Brassica napus","volume":"7","author":"Yang","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_70","doi-asserted-by":"crossref","first-page":"3422","DOI":"10.1038\/s41467-022-30480-8","article-title":"Cas9-induced large deletions and small indels are controlled in a convergent fashion","volume":"13","author":"Kosicki","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_71","doi-asserted-by":"crossref","first-page":"333","DOI":"10.1016\/j.molcel.2021.12.002","article-title":"The CRISPR-Cas toolbox and gene editing technologies","volume":"82","author":"Liu","year":"2022","journal-title":"Mol. Cell"},{"key":"ref_72","doi-asserted-by":"crossref","unstructured":"Yuan, B., Bi, C., Tian, Y., Wang, J., Jin, Y., Alsayegh, K., Tehseen, M., Yi, G., Zhou, X., and Shao, Y. (2024). Modulation of the microhomology-mediated end joining pathway suppresses large deletions and enhances homology-directed repair following CRISPR-Cas9-induced DNA breaks. BMC Biol., 22.","DOI":"10.1186\/s12915-024-01896-z"},{"key":"ref_73","doi-asserted-by":"crossref","unstructured":"Danchik, C., and Casadevall, A. (2021). Role of cell surface hydrophobicity in the pathogenesis of medically-significant fungi. Front. Cell. Infect. Microbiol., 10.","DOI":"10.3389\/fcimb.2020.594973"},{"key":"ref_74","doi-asserted-by":"crossref","first-page":"420","DOI":"10.1038\/s41587-019-0036-z","article-title":"SignalP 5.0 improves signal peptide predictions using deep neural networks","volume":"37","author":"Tsirigos","year":"2019","journal-title":"Nat. Biotechnol."},{"key":"ref_75","doi-asserted-by":"crossref","first-page":"jcs258936","DOI":"10.1242\/jcs.258936","article-title":"Spc1 regulates the signal peptidase-mediated processing of membrane proteins","volume":"134","author":"Yim","year":"2021","journal-title":"J. Cell Sci."},{"key":"ref_76","doi-asserted-by":"crossref","first-page":"e202211035","DOI":"10.1083\/jcb.202211035","article-title":"Spc2 modulates substrate-and cleavage site-selection in the yeast signal peptidase complex","volume":"223","author":"Chung","year":"2024","journal-title":"J. Cell Biol."},{"key":"ref_77","doi-asserted-by":"crossref","first-page":"422","DOI":"10.1016\/j.ejcb.2018.06.003","article-title":"A comprehensive review of signal peptides: Structure, roles, and applications","volume":"97","author":"Owji","year":"2018","journal-title":"Eur. J. Cell Biol."},{"key":"ref_78","doi-asserted-by":"crossref","unstructured":"Mgbeahuruike, A.C., Kovalchuk, A., Chen, H., Ubhayasekera, W., and Asiegbu, F.O. (2013). Evolutionary analysis of hydrophobin gene family in two wood-degrading basidiomycetes, Phlebia brevispora and Heterobasidion annosum sl. BMC Evol. Biol., 13.","DOI":"10.1186\/1471-2148-13-240"},{"key":"ref_79","doi-asserted-by":"crossref","first-page":"15955","DOI":"10.1074\/jbc.M110.214197","article-title":"Recruitment of class I hydrophobins to the air: Water interface initiates a multi-step process of functional amyloid formation","volume":"286","author":"Morris","year":"2011","journal-title":"J. Biol. Chem."},{"key":"ref_80","doi-asserted-by":"crossref","first-page":"4783","DOI":"10.1021\/acs.biomac.3c00542","article-title":"Surface-induced hydrophobin assemblies with versatile properties and distinct underlying structures","volume":"24","author":"Siddiquee","year":"2023","journal-title":"Biomacromolecules"},{"key":"ref_81","doi-asserted-by":"crossref","first-page":"366","DOI":"10.1038\/s41598-021-04223-6","article-title":"The N-terminal tail of the hydrophobin SC16 is not required for rodlet formation","volume":"12","author":"Vergunst","year":"2022","journal-title":"Sci. Rep."},{"key":"ref_82","doi-asserted-by":"crossref","unstructured":"Dort, E.N., Tanguay, P., and Hamelin, R.C. (2020). CRISPR\/Cas9 gene editing: An unexplored frontier for forest pathology. Front. Plant Sci., 11.","DOI":"10.3389\/fpls.2020.01126"},{"key":"ref_83","doi-asserted-by":"crossref","first-page":"15007","DOI":"10.1038\/celldisc.2015.7","article-title":"Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR\/Cas9 system","volume":"1","author":"Liu","year":"2015","journal-title":"Cell Discov."},{"key":"ref_84","doi-asserted-by":"crossref","unstructured":"Paul, N.C., Park, S.-W., Liu, H., Choi, S., Ma, J., MacCready, J.S., Chilvers, M.I., and Sang, H. (2021). Plant and fungal genome editing to enhance plant disease resistance using the CRISPR\/Cas9 system. Front. Plant Sci., 12.","DOI":"10.3389\/fpls.2021.700925"},{"key":"ref_85","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1007\/s00203-021-02723-7","article-title":"Applications of CRISPR\/Cas gene-editing technology in yeast and fungi","volume":"204","author":"Liao","year":"2022","journal-title":"Arch. Microbiol."},{"key":"ref_86","doi-asserted-by":"crossref","first-page":"335","DOI":"10.5511\/plantbiotechnology.24.0709a","article-title":"Improvement of genome editing efficiency by Cas9 codon optimization in Japanese cedar (Cryptomeria japonica D. Don)","volume":"41","author":"Nanasato","year":"2024","journal-title":"Plant Biotechnol."},{"key":"ref_87","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1186\/s10086-022-02033-6","article-title":"Marker-free genome editing in the edible mushroom, Pleurotus ostreatus, using transient expression of genes required for CRISPR\/Cas9 and for selection","volume":"68","author":"Koshi","year":"2022","journal-title":"J. Wood Sci."},{"key":"ref_88","doi-asserted-by":"crossref","first-page":"1697","DOI":"10.1128\/IAI.66.4.1697-1707.1998","article-title":"Electrotransformation and expression of bacterial genes encoding hygromycin phosphotransferase and \u03b2-galactosidase in the pathogenic fungus Histoplasma capsulatum","volume":"66","author":"Woods","year":"1998","journal-title":"Infect. Immun."},{"key":"ref_89","doi-asserted-by":"crossref","unstructured":"N\u00f8dvig, C.S., Nielsen, J.B., Kogle, M.E., and Mortensen, U.H. (2015). A CRISPR-Cas9 system for genetic engineering of filamentous fungi. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0133085"},{"key":"ref_90","doi-asserted-by":"crossref","first-page":"2543","DOI":"10.1002\/bit.25662","article-title":"Tailor-made CRISPR\/Cas system for highly efficient targeted gene replacement in the rice blast fungus","volume":"112","author":"Arazoe","year":"2015","journal-title":"Biotechnol. Bioeng."},{"key":"ref_91","doi-asserted-by":"crossref","first-page":"20469","DOI":"10.1038\/s41598-024-71540-x","article-title":"A user-friendly CRISPR\/Cas9 system for mutagenesis of Neurospora crassa","volume":"14","author":"Kempken","year":"2024","journal-title":"Sci. Rep."},{"key":"ref_92","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1186\/s13059-025-03488-8","article-title":"Genome-wide CRISPR guide RNA design and specificity analysis with GuideScan2","volume":"26","author":"Schmidt","year":"2025","journal-title":"Genome Biol."},{"key":"ref_93","doi-asserted-by":"crossref","first-page":"102697","DOI":"10.1016\/j.copbio.2022.102697","article-title":"SgRNA engineering for improved genome editing and expanded functional assays","volume":"75","author":"Dong","year":"2022","journal-title":"Curr. Opin. Biotechnol."},{"key":"ref_94","doi-asserted-by":"crossref","first-page":"21","DOI":"10.2147\/BTT.S429411","article-title":"Recent advancements in reducing the off-target effect of CRISPR-Cas9 genome editing","volume":"18","author":"Mengstie","year":"2024","journal-title":"Biol. Targets Ther."},{"key":"ref_95","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1080\/19768354.2024.2322054","article-title":"Factors affecting the cleavage efficiency of the CRISPR-Cas9 system","volume":"28","author":"Jung","year":"2024","journal-title":"Anim. Cells Syst."},{"key":"ref_96","doi-asserted-by":"crossref","first-page":"108","DOI":"10.1016\/j.gpb.2022.02.006","article-title":"Computational tools and resources for CRISPR\/Cas genome editing","volume":"21","author":"Li","year":"2023","journal-title":"Genom. Proteom. Bioinform."},{"key":"ref_97","doi-asserted-by":"crossref","first-page":"5175","DOI":"10.1093\/bioinformatics\/btac681","article-title":"Evaluation of efficiency prediction algorithms and development of ensemble model for CRISPR\/Cas9 gRNA selection","volume":"38","author":"Chen","year":"2022","journal-title":"Bioinformatics"},{"key":"ref_98","doi-asserted-by":"crossref","first-page":"76","DOI":"10.1186\/s12934-020-01334-z","article-title":"Disruption or reduced expression of the orotidine-5\u2032-decarboxylase gene pyrG increases citric acid production: A new discovery during recyclable genome editing in Aspergillus niger","volume":"19","author":"Zhang","year":"2020","journal-title":"Microb. Cell Fact."},{"key":"ref_99","doi-asserted-by":"crossref","unstructured":"Rehman, L., Su, X., Guo, H., Qi, X., and Cheng, H. (2016). Protoplast transformation as a potential platform for exploring gene function in Verticillium dahliae. BMC Biotechnol., 16.","DOI":"10.1186\/s12896-016-0287-4"},{"key":"ref_100","doi-asserted-by":"crossref","unstructured":"Schindele, P., Wolter, F., and Puchta, H. (2020). CRISPR guide RNA design guidelines for efficient genome editing. RNA Tagging: Methods and Protocols, Springer.","DOI":"10.1007\/978-1-0716-0712-1_19"},{"key":"ref_101","doi-asserted-by":"crossref","first-page":"765","DOI":"10.1038\/nbt.4192","article-title":"Repair of double-strand breaks induced by CRISPR\u2013Cas9 leads to large deletions and complex rearrangements","volume":"36","author":"Kosicki","year":"2018","journal-title":"Nat. Biotechnol."},{"key":"ref_102","doi-asserted-by":"crossref","first-page":"7402","DOI":"10.1093\/nar\/gkz459","article-title":"Microhomologies are prevalent at Cas9-induced larger deletions","volume":"47","author":"Owens","year":"2019","journal-title":"Nucleic Acids Res."},{"key":"ref_103","doi-asserted-by":"crossref","first-page":"12867","DOI":"10.1038\/s41598-017-12740-6","article-title":"Revealing hidden complexities of genomic rearrangements generated with Cas9","volume":"7","author":"Boroviak","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_104","doi-asserted-by":"crossref","first-page":"699","DOI":"10.1016\/j.molcel.2018.11.031","article-title":"Target-specific precision of CRISPR-mediated genome editing","volume":"73","author":"Chakrabarti","year":"2019","journal-title":"Mol. Cell"},{"key":"ref_105","doi-asserted-by":"crossref","first-page":"1686","DOI":"10.1016\/j.csbj.2020.06.026","article-title":"CRISPR-sub: Analysis of DNA substitution mutations caused by CRISPR-Cas9 in human cells","volume":"18","author":"Hwang","year":"2020","journal-title":"Comput. Struct. Biotechnol. J."},{"key":"ref_106","doi-asserted-by":"crossref","first-page":"351","DOI":"10.1136\/mp.54.5.351","article-title":"PCR amplification introduces errors into mononucleotide and dinucleotide repeat sequences","volume":"54","author":"Clarke","year":"2001","journal-title":"Mol. Pathol."},{"key":"ref_107","first-page":"1000106","article-title":"Accuracy of next generation sequencing platforms","volume":"1","author":"Fox","year":"2014","journal-title":"Next Gener. Seq. Appl."},{"key":"ref_108","doi-asserted-by":"crossref","first-page":"759","DOI":"10.1111\/j.1755-0998.2011.03024.x","article-title":"Field guide to next-generation DNA sequencers","volume":"11","author":"Glenn","year":"2011","journal-title":"Mol. Ecol. Resour."},{"key":"ref_109","doi-asserted-by":"crossref","unstructured":"Shumega, A.R., Pavlov, Y.I., Chirinskaite, A.V., Rubel, A.A., Inge-Vechtomov, S.G., and Stepchenkova, E.I. (2024). CRISPR\/Cas9 as a mutagenic factor. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms25020823"},{"key":"ref_110","doi-asserted-by":"crossref","first-page":"e29061","DOI":"10.1016\/j.heliyon.2024.e29061","article-title":"Unexpected mutations occurred in CRISPR\/Cas9 edited Drosophila analyzed by deeply whole genomic sequencing","volume":"10","author":"Xiao","year":"2024","journal-title":"Heliyon"},{"key":"ref_111","doi-asserted-by":"crossref","unstructured":"Jensen, B.G., Andersen, M.R., Pedersen, M.H., Frisvad, J.C., and S\u00f8ndergaard, I. (2010). Hydrophobins from Aspergillus species cannot be clearly divided into two classes. BMC Res. Notes, 3.","DOI":"10.1186\/1756-0500-3-344"},{"key":"ref_112","first-page":"1575","article-title":"Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea","volume":"5","author":"Talbot","year":"1993","journal-title":"Plant Cell"},{"key":"ref_113","doi-asserted-by":"crossref","first-page":"1224","DOI":"10.1111\/j.1365-2958.2005.04750.x","article-title":"MHP1, a Magnaporthe grisea hydrophobin gene, is required for fungal development and plant colonization","volume":"57","author":"Kim","year":"2005","journal-title":"Mol. Microbiol."},{"key":"ref_114","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1007\/s00294-012-0366-6","article-title":"Hydrophobin genes of the entomopathogenic fungus, Metarhizium brunneum, are differentially expressed and corresponding mutants are decreased in virulence","volume":"58","author":"Sevim","year":"2012","journal-title":"Curr. Genet."},{"key":"ref_115","doi-asserted-by":"crossref","first-page":"931","DOI":"10.1128\/EC.4.5.931-936.2005","article-title":"A hydrophobin of the chestnut blight fungus, Cryphonectria parasitica, is required for stromal pustule eruption","volume":"4","author":"Kazmierczak","year":"2005","journal-title":"Eukaryot. Cell"},{"key":"ref_116","doi-asserted-by":"crossref","first-page":"450","DOI":"10.1094\/MPMI-9-0450","article-title":"MPG1, a gene encoding a fungal hydrophobin of Magnaporthe grisea, is involved in surface recognition","volume":"9","author":"Beckerman","year":"1996","journal-title":"Mol. Plant-Microbe Interact. MPMI"},{"key":"ref_117","doi-asserted-by":"crossref","first-page":"15967","DOI":"10.1073\/pnas.0901477106","article-title":"Genome-wide functional analysis reveals that infection-associated fungal autophagy is necessary for rice blast disease","volume":"106","author":"Kershaw","year":"2009","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_118","doi-asserted-by":"crossref","first-page":"2635","DOI":"10.1111\/mpp.12734","article-title":"Fungal attack and host defence pathways unveiled in near-avirulent interactions of Penicillium expansum creA mutants on apples","volume":"19","author":"Tannous","year":"2018","journal-title":"Mol. Plant Pathol."},{"key":"ref_119","doi-asserted-by":"crossref","first-page":"341","DOI":"10.1038\/s41392-024-02036-3","article-title":"New insights into protein\u2013protein interaction modulators in drug discovery and therapeutic advance","volume":"9","author":"Nada","year":"2024","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_120","doi-asserted-by":"crossref","unstructured":"Sarkar, A., and Roy-Barman, S. (2021). Spray-induced silencing of pathogenicity gene MoDES1 via exogenous double-stranded RNA can confer partial resistance against fungal blast in rice. Front. Plant Sci., 12.","DOI":"10.3389\/fpls.2021.733129"},{"key":"ref_121","doi-asserted-by":"crossref","first-page":"19324","DOI":"10.1073\/pnas.1306373110","article-title":"Host-induced gene silencing of cytochrome P450 lanosterol C14\u03b1-demethylase\u2013encoding genes confers strong resistance to Fusarium species","volume":"110","author":"Koch","year":"2013","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_122","doi-asserted-by":"crossref","unstructured":"Wang, M., and Dean, R.A. (2022). Host induced gene silencing of Magnaporthe oryzae by targeting pathogenicity and development genes to control rice blast disease. Front. Plant Sci., 13.","DOI":"10.3389\/fpls.2022.959641"},{"key":"ref_123","doi-asserted-by":"crossref","first-page":"4","DOI":"10.1016\/j.tim.2016.11.011","article-title":"Spray-induced gene silencing: A powerful innovative strategy for crop protection","volume":"25","author":"Wang","year":"2017","journal-title":"Trends Microbiol."},{"key":"ref_124","doi-asserted-by":"crossref","unstructured":"Bahn, Y.-S. (2015). Exploiting fungal virulence-regulating transcription factors as novel antifungal drug targets. PLoS Pathog., 11.","DOI":"10.1371\/journal.ppat.1004936"},{"key":"ref_125","doi-asserted-by":"crossref","first-page":"1587","DOI":"10.1007\/s00253-014-6319-x","article-title":"Applications of hydrophobins: Current state and perspectives","volume":"99","author":"Scholtmeijer","year":"2015","journal-title":"Appl. Microbiol. Biotechnol."}],"container-title":["Journal of Fungi"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2309-608X\/12\/8\/594\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,11]],"date-time":"2026-08-11T09:02:36Z","timestamp":1786438956000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2309-608X\/12\/8\/594"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,11]]},"references-count":125,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["jof12080594"],"URL":"https:\/\/doi.org\/10.3390\/jof12080594","relation":{},"ISSN":["2309-608X"],"issn-type":[{"value":"2309-608X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,11]]}}}