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It provides plants with rigidity and protection, but its recalcitrant nature also presents a significant barrier to lignocellulose valorization. The white-rot fungus\n                      <jats:italic toggle=\"yes\">Phanerochaete chrysosporium<\/jats:italic>\n                      is among nature\u2019s most efficient lignin degraders, and its ligninolytic capabilities have been subjected to intensive investigations. Genome editing with precision is crucial for elucidating the\n                      <jats:italic toggle=\"yes\">in vivo<\/jats:italic>\n                      mechanisms of its ligninolytic actions, but genetic manipulations of\n                      <jats:italic toggle=\"yes\">P. chrysosporium<\/jats:italic>\n                      are often plagued by imprecision. This technical nuisance is driven primarily by canonical non-homologous end joining (c-NHEJ), a DNA repair system that requires little homology and depends on the binding of the Ku70\/Ku80 heterodimer to double-strand break (DSB) ends. Loss of Ku70 or Ku80 abolishes c-NHEJ and significantly improves genome editing precision in many filamentous fungi, but it has yet to be examined and exploited in\n                      <jats:italic toggle=\"yes\">P. chrysosporium<\/jats:italic>\n                      . Here, we constructed a homozygous\n                      <jats:italic toggle=\"yes\">ku70<\/jats:italic>\n                      \u0394 mutant in a meiotic homokaryon of clear genetic background. Loss of Ku70 minimally impacts growth but significantly increases homologous recombination frequency from ~2% to ~66%, with ~32% of the latter being homozygous. Unexpectedly, loss of Ku70 also promotes mononucleate conidiation, which may facilitate isolation of homozygous mutants. Taken together, our work provides a valuable genetic tool to understand and exploit\n                      <jats:italic toggle=\"yes\">P. chrysosporium<\/jats:italic>\n                      \u2019s remarkable ligninolytic capabilities.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Genome editing with precision is essential to unraveling the intricacies of\n                        <jats:italic toggle=\"yes\">P. chrysosporium<\/jats:italic>\n                        \u2019s exceptional ligninolytic capabilities, but the available tools are generally imprecise due to the dominance of non-homologous recombination, a problem that is further exacerbated by the discontinuation of Novozyme 234. We tackle these challenges by reestablishing protoplast-based transformation with Lywallzyme as an alternative. Importantly, we demonstrate that inactivation of c-NHEJ by deleting\n                        <jats:italic toggle=\"yes\">ku70<\/jats:italic>\n                        significantly increases gene knockout efficiency and report the unexpected involvement of c-NHEJ in regulating the number of nuclei during conidiation. Our work paves the way for future ventures into understanding ligninolysis in\n                        <jats:italic toggle=\"yes\">P. chrysosporium<\/jats:italic>\n                        and building superior chassis for industrial applications.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/aem.00497-26","type":"journal-article","created":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T13:01:16Z","timestamp":1789390876000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Loss of Ku70 promotes mononucleate conidiation and homologous recombination in\n                    <i>Phanerochaete chrysosporium<\/i>"],"prefix":"10.1128","author":[{"given":"Hao","family":"Wu","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/018rbtf37","id-type":"ROR","asserted-by":"publisher"}],"name":"College of Biotechnology, Tianjin University of Science and Technology","place":["Tianjin, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiaojiao","family":"Zhang","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/042pyga86","id-type":"ROR","asserted-by":"publisher"}],"name":"In Vitro Synthetic Biology Center, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences","place":["Tianjin, China"]},{"name":"National Center of Technology Innovation For Synthetic Biology","place":["Tianjin, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yi","family":"Zhao","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/018rbtf37","id-type":"ROR","asserted-by":"publisher"}],"name":"College of Biotechnology, Tianjin University of Science and Technology","place":["Tianjin, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lan","family":"Yao","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04v3ywz14","id-type":"ROR","asserted-by":"publisher"}],"name":"College of Food Science and Nutritional Engineering, China Agricultural University","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2488-8636","authenticated-orcid":true,"given":"Aiqun","family":"Yu","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/018rbtf37","id-type":"ROR","asserted-by":"publisher"}],"name":"State Key Laboratory of Food Nutrition and Safety, Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology","place":["Tianjin, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4010-2250","authenticated-orcid":true,"given":"Yi-Heng P. 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