{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T23:34:57Z","timestamp":1790379297629,"version":"4.1.0"},"posted":{"date-parts":[[2024,9,19]]},"group-title":"Genetics","reference-count":66,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2024,9,19]],"date-time":"2024-09-19T00:00:00Z","timestamp":1726704000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"name":"NIH","award":["R21HD120879"],"award-info":[{"award-number":["R21HD120879"]}]},{"name":"NIH","award":["R35GM136644"],"award-info":[{"award-number":["R35GM136644"]}]},{"name":"NIH","award":["T32GM148383-01"],"award-info":[{"award-number":["T32GM148383-01"]}]},{"DOI":"10.13039\/501100000024","name":"Canadian Institutes of Health Research","doi-asserted-by":"crossref","award":["DFD-181599"],"award-info":[{"award-number":["DFD-181599"]}],"id":[{"id":"10.13039\/501100000024","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,11,22]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  The application of CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) technologies in zebrafish has the potential to expand its capacity for the study of gene function significantly. We report proof-of-principle data evaluating transient expression of a codon optimized CRISPRi\/a system for zebrafish across established pigmentary and growth phenotypes. A codon-optimized and catalytically inactive\n                  <jats:italic>cas9<\/jats:italic>\n                  gene (\n                  <jats:italic>dcas9<\/jats:italic>\n                  ) was cloned upstream of codon-optimized Kr\u00fcppel associated box (KRAB) and methyl-CpG binding protein 2 (MeCP2) for CRISPRi, and VP64 for CRISPRa. To validate CRISPRi, we targeted key genes in melanocyte differentiation (\n                  <jats:italic>sox10<\/jats:italic>\n                  and\n                  <jats:italic>mitfa)<\/jats:italic>\n                  ; and melanin production (tyrosinase;\n                  <jats:italic>tyr<\/jats:italic>\n                  ). Microinjection of CRISPRi mRNA and single guide RNAs (sgRNAs) targeting the\n                  <jats:italic>tyr<\/jats:italic>\n                  promoter or 5\u2019-UTR resulted in larvae with hypopigmented epidermal melanocytes. Similarly, CRISPRi targeting of the\n                  <jats:italic>sox10 or mitfa<\/jats:italic>\n                  promoters results in hypopigmentation of epidermal melanocytes consistent with their roles upstream of\n                  <jats:italic>tyr,<\/jats:italic>\n                  and the role of\n                  <jats:italic>sox10<\/jats:italic>\n                  in activation of\n                  <jats:italic>mitfa<\/jats:italic>\n                  . Finally, we tested both CRISPRi\/a to modulate a single gene to yield hypomorphic and hypermorphic effects, selecting\n                  <jats:italic>mrap2a<\/jats:italic>\n                  as our target. This gene regulates energy homeostasis and somatic growth via inhibition of the melanocortin 4 receptor gene (\n                  <jats:italic>mc4r<\/jats:italic>\n                  ). We show that inactivating or activating\n                  <jats:italic>mrap2a<\/jats:italic>\n                  with CRISPRi\/a significantly decreases or increases larval body length, respectively. We demonstrate that CRISPRi\/a can modulate control of zebrafish gene expression, facilitating efficient assay of candidate gene function and disease relevance.\n                <\/jats:p>","DOI":"10.1101\/2024.09.16.613289","type":"posted-content","created":{"date-parts":[[2024,9,20]],"date-time":"2024-09-20T00:30:33Z","timestamp":1726792233000},"source":"Crossref","is-referenced-by-count":3,"title":["Proof of concept study deploying CRISPR inhibition and activation opens new avenues for systematic biological exploration in zebrafish"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8971-3784","authenticated-orcid":false,"given":"Nelson B.","family":"Barrientos","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-6254-0503","authenticated-orcid":false,"given":"Elyse A.","family":"Shoppell","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6544-8541","authenticated-orcid":false,"given":"Rachel J.","family":"Boyd","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Valeria C.","family":"Culotta","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0016-8466","authenticated-orcid":false,"given":"Andrew S.","family":"McCallion","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025112503400769000_2024.09.16.613289v3.1","doi-asserted-by":"crossref","unstructured":"Giffen KP , Liu H , Kramer KL , He DZ . 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