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However, a lack of gene-editing tools appropriated for use in reef fishes has meant the genetic underpinnings for many of their unique traits remain to be investigated. One iconic group of reef fishes ideal for applying this technique are anemonefishes (Amphiprioninae) as they are widely studied for their symbiosis with anemones, sequential hermaphroditism, complex social hierarchies, skin pattern development, and vision, and are raised relatively easily in aquaria. In this study, we developed a gene-editing protocol for applying the CRISPR\/Cas9 system in the false clown anemonefish,<jats:italic>Amphiprion ocellaris<\/jats:italic>. Microinjection of zygotes was used to demonstrate the successful use of our CRISPR\/Cas9 approach at two separate target sites: the rhodopsin-like 2B opsin encoding gene (<jats:italic>RH2B<\/jats:italic>) involved in vision, and Tyrosinase-producing gene (<jats:italic>tyr<\/jats:italic>) involved in the production of melanin. Analysis of the sequenced target gene regions in<jats:italic>A<\/jats:italic>.<jats:italic>ocellaris<\/jats:italic>embryos showed that uptake was as high as 73.3% of injected embryos. Further analysis of the subcloned mutant gene sequences combined with amplicon shotgun sequencing revealed that our approach had a 75% to 100% efficiency in producing biallelic mutations in F0<jats:italic>A<\/jats:italic>.<jats:italic>ocellaris<\/jats:italic>embryos. Moreover, we clearly show a loss-of-function in<jats:italic>tyr<\/jats:italic>mutant embryos which exhibited typical hypomelanistic phenotypes. This protocol is intended as a useful starting point to further explore the potential application of CRISPR\/Cas9 in<jats:italic>A<\/jats:italic>.<jats:italic>ocellaris<\/jats:italic>, as a platform for studying gene function in anemonefishes and other reef fishes.<\/jats:p>","DOI":"10.1371\/journal.pone.0261331","type":"journal-article","created":{"date-parts":[[2021,12,15]],"date-time":"2021-12-15T18:36:52Z","timestamp":1639593412000},"page":"e0261331","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":28,"title":["CRISPR\/Cas9-mediated generation of biallelic F0 anemonefish (Amphiprion ocellaris) mutants"],"prefix":"10.1371","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4967-4113","authenticated-orcid":true,"given":"Laurie J.","family":"Mitchell","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Valerio","family":"Tettamanti","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Justin S.","family":"Rhodes","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"N. 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