{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T18:33:41Z","timestamp":1791311621190,"version":"4.3.1"},"reference-count":15,"publisher":"American Society for Microbiology","issue":"11","license":[{"start":{"date-parts":[[2014,11,1]],"date-time":"2014-11-01T00:00:00Z","timestamp":1414800000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Eukaryot Cell"],"published-print":{"date-parts":[[2014,11]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n          <jats:p>\n            The clustered regularly interspaced short palindromic repeat (CRISPR)\/Cas9 system has become a powerful and precise tool for targeted gene modification (e.g., gene knockout and gene replacement) in numerous eukaryotic organisms. Initial attempts to apply this technology to a model, the single-cell alga,\n            <jats:named-content content-type=\"genus-species\">Chlamydomonas reinhardtii<\/jats:named-content>\n            , failed to yield cells containing edited genes. To determine if the Cas9 and single guide RNA (sgRNA) genes were functional in\n            <jats:named-content content-type=\"genus-species\">C. reinhardtii<\/jats:named-content>\n            , we tested the ability of a codon-optimized Cas9 gene along with one of four different sgRNAs to cause targeted gene disruption during a 24-h period immediately following transformation. All three exogenously supplied gene targets as well as the endogenous\n            <jats:italic>FKB12<\/jats:italic>\n            (rapamycin sensitivity) gene of\n            <jats:named-content content-type=\"genus-species\">C. reinhardtii<\/jats:named-content>\n            displayed distinct Cas9\/sgRNA-mediated target site modifications as determined by DNA sequencing of cloned PCR amplicons of the target site region. Success in transient expression of Cas9 and sgRNA genes contrasted with the recovery of only a single rapamycin-resistant colony bearing an appropriately modified\n            <jats:italic>FKB12<\/jats:italic>\n            target site in 16 independent transformation experiments involving &gt;10\n            <jats:sup>9<\/jats:sup>\n            cells. Failure to recover transformants with intact or expressed Cas9 genes following transformation with the Cas9 gene alone (or even with a gene encoding a Cas9 lacking nuclease activity) provided strong suggestive evidence for Cas9 toxicity when Cas9 is produced constitutively in\n            <jats:named-content content-type=\"genus-species\">C. reinhardtii<\/jats:named-content>\n            . The present results provide compelling evidence that Cas9 and sgRNA genes function properly in\n            <jats:named-content content-type=\"genus-species\">C. reinhardtii<\/jats:named-content>\n            to cause targeted gene modifications and point to the need for a focus on development of methods to properly stem Cas9 production and\/or activity following gene editing.\n          <\/jats:p>","DOI":"10.1128\/ec.00213-14","type":"journal-article","created":{"date-parts":[[2014,9,20]],"date-time":"2014-09-20T00:54:25Z","timestamp":1411174465000},"page":"1465-1469","update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":335,"title":["Successful Transient Expression of Cas9 and Single Guide RNA Genes in Chlamydomonas reinhardtii"],"prefix":"10.1128","volume":"13","author":[{"given":"Wenzhi","family":"Jiang","sequence":"first","affiliation":[{"name":"Department of Biochemistry, University of Nebraska, Lincoln, Nebraska, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrew J.","family":"Brueggeman","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, University of Nebraska, Lincoln, Nebraska, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kempton M.","family":"Horken","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, University of Nebraska, Lincoln, Nebraska, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas M.","family":"Plucinak","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, University of Nebraska, Lincoln, Nebraska, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Donald P.","family":"Weeks","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, University of Nebraska, Lincoln, Nebraska, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_3_2_2","doi-asserted-by":"publisher","DOI":"10.1105\/tpc.114.124099"},{"key":"e_1_3_3_3_2","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.2649"},{"key":"e_1_3_3_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2014.05.010"},{"key":"e_1_3_3_5_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkt780"},{"key":"e_1_3_3_6_2","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.2654"},{"key":"e_1_3_3_7_2","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.2655"},{"key":"e_1_3_3_8_2","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.2650"},{"key":"e_1_3_3_9_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1400822111"},{"key":"e_1_3_3_10_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0099225"},{"key":"e_1_3_3_11_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gku806"},{"key":"e_1_3_3_12_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.1225829"},{"key":"e_1_3_3_13_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.1232033"},{"key":"e_1_3_3_14_2","volume-title":"The Chlamydomonas sourcebook: a comprehensive guide to biology and laboratory use","author":"Harris EH","year":"1989","unstructured":"HarrisEH. 1989. The Chlamydomonas sourcebook: a comprehensive guide to biology and laboratory use. Academic Press, San Diego, CA."},{"key":"e_1_3_3_15_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2013.06.044"},{"key":"e_1_3_3_16_2","doi-asserted-by":"publisher","DOI":"10.1104\/pp.105.070847"}],"container-title":["Eukaryotic Cell"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/EC.00213-14","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/EC.00213-14","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,2,23]],"date-time":"2022-02-23T10:02:37Z","timestamp":1645610557000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.asm.org\/doi\/10.1128\/EC.00213-14"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2014,11]]},"references-count":15,"journal-issue":{"issue":"11","published-print":{"date-parts":[[2014,11]]}},"alternative-id":["10.1128\/EC.00213-14"],"URL":"https:\/\/doi.org\/10.1128\/ec.00213-14","relation":{},"ISSN":["1535-9778","1535-9786"],"issn-type":[{"value":"1535-9778","type":"print"},{"value":"1535-9786","type":"electronic"}],"subject":[],"published":{"date-parts":[[2014,11]]},"assertion":[{"value":"2014-09-15","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2014-09-16","order":1,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2014-10-29","order":2,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}]}}