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Protocols for CRISPR-Cas9 gene editing have already been developed for Phytophthoras, an important group of oomycete plant pathogens; however, these efforts have exclusively focused on agricultural pathosystems, with research lacking for forest pathosystems. We sought to develop CRISPR-Cas9 gene editing in two forest pathogenic Phytophthoras,\n              <jats:italic>Phytophthora cactorum<\/jats:italic>\n              and\n              <jats:italic>P. ramorum<\/jats:italic>\n              , using a plasmid-ribonucleoprotein (RNP) co-transformation approach. Our gene target in both species was the ortholog of\n              <jats:italic>PcORP1<\/jats:italic>\n              , which encodes an oxysterol-binding protein that is the target of the fungicide oxathiapiprolin in the agricultural pathogen\n              <jats:italic>P. capsici<\/jats:italic>\n              . We delivered liposome complexes, each containing plasmid DNA and CRISPR-Cas9 RNPs, to\n              <jats:italic>Phytophthora<\/jats:italic>\n              protoplasts using a polyethylene glycol-mediated transformation protocol. We obtained two\n              <jats:italic>ORP1<\/jats:italic>\n              mutants in\n              <jats:italic>P. cactorum<\/jats:italic>\n              but were unable to obtain any mutants in\n              <jats:italic>P. ramorum<\/jats:italic>\n              . The two\n              <jats:italic>P. cactorum<\/jats:italic>\n              mutants exhibited decreased resistance to oxathiapiprolin, as measured by their radial growth relative to wild-type cultures on oxathiapiprolin-supplemented medium. Our results demonstrate the potential for RNP-mediated CRISPR-Cas9 gene editing in\n              <jats:italic>P. cactorum<\/jats:italic>\n              and provide a foundation for future optimization of our protocol in other forest pathogenic\n              <jats:italic>Phytophthora<\/jats:italic>\n              species.\n            <\/jats:p>\n            <jats:sec>\n              <jats:title>IMPORTANCE<\/jats:title>\n              <jats:p>\n                CRISPR-Cas9 gene editing has become a valuable tool for characterizing the genetics driving virulence and pathogenicity in plant pathogens. CRISPR-Cas9 protocols are now well-established in several\n                <jats:italic>Phytophthora<\/jats:italic>\n                species, an oomycete genus with significant economic and ecological impact globally. These protocols, however, have been developed for agricultural\n                <jats:italic>Phytophthora<\/jats:italic>\n                pathogens only; CRISPR-Cas9 systems have not yet been developed for any forest pathogenic Phytophthoras. In this study, we sought to establish CRISPR-Cas9 gene editing in two forest\n                <jats:italic>Phytophthora<\/jats:italic>\n                pathogens that cause widespread tree mortality:\n                <jats:italic>P. cactorum<\/jats:italic>\n                and\n                <jats:italic>P. ramorum<\/jats:italic>\n                . We successfully obtained gene mutations in\n                <jats:italic>P. cactorum<\/jats:italic>\n                and demonstrated a decrease in fungicide resistance, a trait that could impact the pathogen\u2019s ability to cause disease. However, the same protocol did not yield any mutants in\n                <jats:italic>P. ramorum<\/jats:italic>\n                . The results of our study will serve as a baseline for the development of CRISPR-Cas9 gene editing in forest Phytophthoras and other oomycetes.\n              <\/jats:p>\n            <\/jats:sec>\n          <\/jats:sec>","DOI":"10.1128\/spectrum.03012-24","type":"journal-article","created":{"date-parts":[[2025,2,27]],"date-time":"2025-02-27T15:25:11Z","timestamp":1740669911000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":2,"title":["Novel application of ribonucleoprotein-mediated CRISPR-Cas9 gene editing in plant pathogenic oomycete species"],"prefix":"10.1128","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7814-1060","authenticated-orcid":true,"given":"Erika N.","family":"Dort","sequence":"first","affiliation":[{"name":"Department of Forest & Conservation Sciences, Faculty of Forestry, University of British Columbia","place":["Vancouver, Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5925-9867","authenticated-orcid":true,"given":"Nicolas","family":"Feau","sequence":"additional","affiliation":[{"name":"Pacific Forestry Centre, Canadian Forest Service, Natural Resources Canada","place":["Victoria, Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Richard C.","family":"Hamelin","sequence":"additional","affiliation":[{"name":"Department of Forest & Conservation Sciences, Faculty of Forestry, University of British Columbia","place":["Vancouver, Canada"]},{"name":"Institut de Biologie Int\u00e9grative et des Syst\u00e8mes (IBIS), Universit\u00e9 Laval","place":["Qu\u00e9bec, Canada"]},{"name":"D\u00e9partement des Sciences du bois et de la For\u00eat, Facult\u00e9 de Foresterie et G\u00e9ographie, Universit\u00e9 Laval","place":["Qu\u00e9bec, Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_3_2_2","first-page":"75","volume-title":"Die gegenw\u00e4rtig herrschende Kartoffelkrankenheit, ihre Ursache und ihre Verh\u00fctung (The current prevailing potato disease, its cause and treatment)","author":"de Bary A","year":"1861","unstructured":"de Bary A. 1861. 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