{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T21:25:51Z","timestamp":1792790751653,"version":"4.5.0"},"reference-count":25,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2020,12,3]],"date-time":"2020-12-03T00:00:00Z","timestamp":1606953600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2020,12,3]],"date-time":"2020-12-03T00:00:00Z","timestamp":1606953600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"name":"Faculty of Medicine at the University of Helsinki","id":[{"id":"https:\/\/ror.org\/040af2s02","id-type":"ROR","asserted-by":"crossref"}]},{"name":"The Tampere Graduate Program in Biomedicine and Biotechnology"},{"DOI":"10.13039\/501100002341","name":"Academy of Finland","doi-asserted-by":"publisher","award":["297727"],"award-info":[{"award-number":["297727"]}],"id":[{"id":"10.13039\/501100002341","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Sigrid Juselius Foundation","id":[{"id":"https:\/\/ror.org\/00ckakm23","id-type":"ROR","asserted-by":"crossref"}]},{"DOI":"10.13039\/100015735","name":"Helsinki Institute of Life Science, Helsingin Yliopisto","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100015735","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Fellow grant and European Research Council","award":["724922"],"award-info":[{"award-number":["724922"]}],"id":[{"id":"https:\/\/ror.org\/0472cxd90","id-type":"ROR","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100004047","name":"Karolinska Institute","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100004047","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    CRISPR\/Cas9 driven mutagenesis in zygotes is a popular tool for introducing targeted mutations in model organisms. Compared to mouse, mutagenesis in zebrafish is relatively inefficient and results in somatic mosaicism most likely due to a short single-cell stage of about 40\u00a0min. Here we explored two options to improve CRISPR\/Cas9 mutagenesis in zebrafish\u2014extending the single-cell stage and defining conditions for carrying out mutagenesis in oocytes prior to in vitro fertilization. Previous work has shown that ovarian fluid from North American salmon species (coho and chinook salmon) prolong oocyte survival ex vivo so that they are viable for hours instead of dying within minutes if left untreated. We found that commonly farmed rainbow trout (\n                    <jats:italic>Oncorhynchus mykiss<\/jats:italic>\n                    ) ovarian fluid (RTOF) has similar effect on zebrafish oocyte viability. In order to prolong single-cell stage, we incubated zebrafish zygotes in hydrogen sulfide (H\n                    <jats:sub>2<\/jats:sub>\n                    S) and RTOF but failed to see any effect. However, the reduction of temperature from standard 28 to 12\u00a0\u00b0C postponed the first cell division by about an hour. In addition, the reduction in temperature was associated with increased CRISPR\/Cas9 mutagenesis rate. These results suggest that the easily applicable reduction in temperature facilitates CRISPR\/Cas9 mutagenesis in zebrafish.\n                  <\/jats:p>","DOI":"10.1038\/s41598-020-77677-9","type":"journal-article","created":{"date-parts":[[2020,12,3]],"date-time":"2020-12-03T06:04:47Z","timestamp":1606975487000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Improving CRISPR\/Cas9 mutagenesis efficiency by delaying the early development of zebrafish embryos"],"prefix":"10.1038","volume":"10","author":[{"given":"M.","family":"Terzioglu","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"A.","family":"Saralahti","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"H.","family":"Piippo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"M.","family":"R\u00e4met","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"J.-O.","family":"Andressoo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2020,12,3]]},"reference":[{"key":"77677_CR1","doi-asserted-by":"publisher","first-page":"293","DOI":"10.1038\/291293a0","volume":"291","author":"G Streisinger","year":"1981","unstructured":"Streisinger, G., Walker, C., Dower, N., Knauber, D. & Singer, F. Production of clones of homozygous diploid zebra fish (Brachydanio rerio). Nature 291, 293\u2013296 (1981).","journal-title":"Nature"},{"key":"77677_CR2","doi-asserted-by":"publisher","first-page":"498","DOI":"10.1038\/nature12111","volume":"496","author":"K Howe","year":"2013","unstructured":"Howe, K. et al. The zebrafish reference genome sequence and its relationship to the human genome. Nature 496, 498\u2013503 (2013).","journal-title":"Nature"},{"key":"77677_CR3","doi-asserted-by":"publisher","first-page":"3103","DOI":"10.1242\/dev.112003","volume":"141","author":"S Schulte-Merker","year":"2014","unstructured":"Schulte-Merker, S. & Stainier, D. Y. R. Out with the old, in with the new: Reassessing morpholino knockdowns in light of genome editing technology. Development. 141, 3103\u20133104 (2014).","journal-title":"Development."},{"key":"77677_CR4","doi-asserted-by":"publisher","first-page":"227","DOI":"10.1038\/nbt.2501","volume":"31","author":"WY Hwang","year":"2013","unstructured":"Hwang, W. Y. et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat. Biotechnol. 31, 227\u2013229 (2013).","journal-title":"Nat. Biotechnol."},{"key":"77677_CR5","doi-asserted-by":"publisher","first-page":"13904","DOI":"10.1073\/pnas.1308335110","volume":"110","author":"LE Jao","year":"2013","unstructured":"Jao, L. E., Wente, S. R. & Chen, W. Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. PNAS 110, 13904\u201313909 (2013).","journal-title":"PNAS"},{"key":"77677_CR6","doi-asserted-by":"publisher","first-page":"535","DOI":"10.1038\/nmeth.3360","volume":"12","author":"AN Shah","year":"2015","unstructured":"Shah, A. N., Davey, C. F., Whitebirch, A. C., Miller, A. C. & Moens, C. B. Rapid reverse genetic screening using CRISPR in zebrafish. Nat. Methods. 12, 535\u2013540 (2015).","journal-title":"Nat. Methods."},{"key":"77677_CR7","doi-asserted-by":"publisher","first-page":"1030","DOI":"10.1101\/gr.186379.114","volume":"25","author":"GK Varshney","year":"2015","unstructured":"Varshney, G. K. et al. High-throughput gene targeting and phenotyping in zebrafish using CRISPR\/Cas9. Genome. Res. 25, 1030\u20131042 (2015).","journal-title":"Genome. Res."},{"key":"77677_CR8","doi-asserted-by":"publisher","first-page":"156","DOI":"10.1016\/j.ydbio.2018.10.008","volume":"445","author":"M Mehravar","year":"2019","unstructured":"Mehravar, M., Shirazi, A., Nazari, M. & Banan, M. Mosaicism in CRISPR\/Cas9-mediated genome editing. Dev. Biol. 445, 156\u2013162 (2019).","journal-title":"Dev. 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Reproduction 135, 285\u2013292 (2008).","journal-title":"Reproduction"},{"key":"77677_CR17","doi-asserted-by":"publisher","first-page":"44","DOI":"10.1002\/dvdy.23894","volume":"242","author":"S Nair","year":"2013","unstructured":"Nair, S., Lindeman, R. E. & Pelergri, F. In vitro oocyte culture-based manipulation of zebrafish maternal genes. Dev. Dyn. 242, 44\u201352 (2013).","journal-title":"Dev. Dyn."},{"key":"77677_CR18","doi-asserted-by":"publisher","first-page":"518","DOI":"10.1126\/science.1108581","volume":"308","author":"E Blackstone","year":"2005","unstructured":"Blackstone, E., Morrison, M. & Roth, M. B. H2S induces a suspended animation like state in mice. Science 308, 518 (2005).","journal-title":"Science"},{"key":"77677_CR19","doi-asserted-by":"publisher","first-page":"3075","DOI":"10.1113\/jphysiol.2014.271098","volume":"592","author":"CS Porteus","year":"2014","unstructured":"Porteus, C. S. et al. 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