{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,11,5]],"date-time":"2026-11-05T16:22:53Z","timestamp":1793895773607,"version":"4.8.0"},"reference-count":23,"publisher":"Oxford University Press (OUP)","issue":"8","license":[{"start":{"date-parts":[[2025,6,3]],"date-time":"2025-06-03T00:00:00Z","timestamp":1748908800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2025,8,6]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>We recently described CRISPR\/Cas9-based short homology-dependent genome engineering in the human fungal pathogen Cryptococcus neoformans, a haploid budding yeast that is the most common cause of fungal meningitis and an emerging model organism. This was achieved by electroporation of strains stably expressing a codon-optimized Cas9 with 2 separate DNA molecules, one encoding a selectable marker flanked by short homology arms and a second encoding a sgRNA under the control of the U6 snRNA promoter. However, the efficiency of desired homology-dependent repair relative to undesired non-homologous end-joining (NHEJ) events can be low and variable. Here, we describe methods and strains enabling extremely efficient (\u223c99%) homology-dependent genome editing in C. neoformans. This high-efficiency method requires 2 manipulations. First, we placed the sgRNA-expressing segment into the marker-containing DNA flanked by targeting homology; thus, only a single DNA molecule is introduced into cells. Second, we used a strain mutant for the non-homologous end-joining factor Ku80 (encoded by YKU80). We also report the engineering of a yku80::amdS mutant strain harboring an insertion mutation that can be removed scarlessly via recombination between direct repeats. This enables the functional restoration of YKU80 after homology-dependent genome editing after selection against the amdS marker using fluoroacetamide. This approach minimizes documented drawbacks of using Ku-defective strains in downstream experiments. Finally, we describe a plasmid series that enables rapid cloning of sgRNA-marker constructs for genomic manipulation of C. neoformans, including gene deletion and C-terminal tagging. These methods, strains, and plasmids accelerate the genomic manipulation of C. neoformans.<\/jats:p>","DOI":"10.1093\/g3journal\/jkaf118","type":"journal-article","created":{"date-parts":[[2025,6,3]],"date-time":"2025-06-03T15:05:25Z","timestamp":1748963125000},"source":"Crossref","is-referenced-by-count":7,"title":["Near 100% efficient homology-dependent genome engineering in the human fungal pathogen\n                    <i>Cryptococcus neoformans<\/i>"],"prefix":"10.1093","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9462-3297","authenticated-orcid":false,"given":"Matthew J","family":"Nalley","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Biophysics, University of California, San Francisco , 600 16th St., San Francisco, CA 94158 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3577-6218","authenticated-orcid":false,"given":"Sanjita","family":"Banerjee","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Biophysics, University of California, San Francisco , 600 16th St., San Francisco, CA 94158 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6045-9698","authenticated-orcid":false,"given":"Manning Y","family":"Huang","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Biophysics, University of California, San Francisco , 600 16th St., San Francisco, CA 94158 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7400-6657","authenticated-orcid":false,"given":"Hiten D","family":"Madhani","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Biophysics, University of California, San Francisco , 600 16th St., San Francisco, CA 94158 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2025,6,3]]},"reference":[{"issue":"10","key":"2025081214114274700_jkaf118-B1","doi-asserted-by":"publisher","first-page":"e0164322","DOI":"10.1371\/journal.pone.0164322","article-title":"Targeted genome editing via CRISPR in the pathogen Cryptococcus neoformans","volume":"11","author":"Arras","year":"2016","journal-title":"PLoS One"},{"issue":"9","key":"2025081214114274700_jkaf118-B2","doi-asserted-by":"publisher","first-page":"e0163049","DOI":"10.1371\/journal.pone.0163049","article-title":"Chemical inhibitors of non-homologous end joining increase targeted construct integration in Cryptococcus neoformans","volume":"11","author":"Arras","year":"2016","journal-title":"PLoS 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