title: Functional visualization and disruption of targeted genes using CRISPR/Cas9-mediated eGFP reporter integration in zebrafish.
abstract: The CRISPR/Cas9 complex, which is composed of a guide RNA (gRNA) and the Cas9 nuclease, is useful for carrying out genome modifications in various organisms. Recently, the CRISPR/Cas9-mediated locus-specific integration of a reporter, which contains the Mbait sequence targeted using Mbait-gRNA, the hsp70 promoter and the eGFP gene, has allowed the visualization of the target gene expression. However, it has not been ascertained whether the reporter integrations at both targeted alleles cause loss-of-function phenotypes in zebrafish. In this study, we have inserted the Mbait-hs-eGFP reporter into the pax2a gene because the disruption of pax2a causes the loss of the midbrain-hindbrain boundary (MHB) in zebrafish. In the heterozygous Tg[pax2a-hs:eGFP] embryos, MHB formed normally and the eGFP expression recapitulated the endogenous pax2a expression, including the MHB. We observed the loss of the MHB in homozygous Tg[pax2a-hs:eGFP] embryos. Furthermore, we succeeded in integrating the Mbait-hs-eGFP reporter into an uncharacterized gene epdr1. The eGFP expression in heterozygous Tg[epdr1-hs:eGFP] embryos overlapped the epdr1 expression, whereas the distribution of eGFP-positive cells was disorganized in the MHB of homozygous Tg[epdr1-hs:eGFP] embryos. We propose that the locus-specific integration of the Mbait-hs-eGFP reporter is a powerful method to investigate both gene expression profiles and loss-of-function phenotypes.
authors.affiliation: Laboratory for Developmental Biology, Center for Medical Education and Sciences, Graduate School of Medical Science, University of Yamanashi, Shimokato 1110, Chuo, Yamanashi, 409-3898, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Ota
authors.affiliation: Laboratory for Developmental Biology, Center for Medical Education and Sciences, Graduate School of Medical Science, University of Yamanashi, Shimokato 1110, Chuo, Yamanashi, 409-3898, Japan.
authors.forename: Kiyohito
authors.initials: K
authors.lastname: Taimatsu
authors.affiliation: Laboratory for Developmental Biology, Center for Medical Education and Sciences, Graduate School of Medical Science, University of Yamanashi, Shimokato 1110, Chuo, Yamanashi, 409-3898, Japan.
authors.forename: Kanoko
authors.initials: K
authors.lastname: Yanagi
authors.affiliation: Laboratory for Developmental Biology, Center for Medical Education and Sciences, Graduate School of Medical Science, University of Yamanashi, Shimokato 1110, Chuo, Yamanashi, 409-3898, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Namiki
authors.affiliation: Laboratory for Developmental Biology, Center for Medical Education and Sciences, Graduate School of Medical Science, University of Yamanashi, Shimokato 1110, Chuo, Yamanashi, 409-3898, Japan.
authors.forename: Rie
authors.initials: R
authors.lastname: Ohga
authors.affiliation: National Institutes of Natural Sciences, Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, Okazaki, Aichi, 444-8787, Japan.
authors.forename: Shin-Ichi
authors.initials: SI
authors.lastname: Higashijima
authors.affiliation: Laboratory for Developmental Biology, Center for Medical Education and Sciences, Graduate School of Medical Science, University of Yamanashi, Shimokato 1110, Chuo, Yamanashi, 409-3898, Japan.
authors.forename: Atsuo
authors.initials: A
authors.lastname: Kawahara
mesh_terms: D000483:Alleles; D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics; D064113:CRISPR-Cas Systems / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics*; D016678:Genome / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics*; D008636:Mesencephalon / Q000378:metabolism; D051762:PAX2 Transcription Factor / Q000235:genetics; D010641:Phenotype; D011401:Promoter Regions, Genetic / Q000235:genetics; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D012249:Rhombencephalon / Q000378:metabolism; D015027:Zebrafish / Q000235:genetics*; D029961:Zebrafish Proteins / Q000235:genetics*
chemical_list: D051762:PAX2 Transcription Factor; D000094704:RNA, Guide, CRISPR-Cas Systems; D029961:Zebrafish Proteins; D049452:Green Fluorescent Proteins