title: Efficient Multiple Genome Modifications Induced by the crRNAs, tracrRNA and Cas9 Protein Complex in Zebrafish.
abstract: The type II clustered regularly interspaced short palindromic repeats (CRISPR) associated with Cas9 endonuclease (CRISPR/Cas9) has become a powerful genetic tool for understanding the function of a gene of interest. In zebrafish, the injection of Cas9 mRNA and guide-RNA (gRNA), which are prepared using an in vitro transcription system, efficiently induce DNA double-strand breaks (DSBs) at the targeted genomic locus. Because gRNA was originally constructed by fusing two short RNAs CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), we examined the effect of synthetic crRNAs and tracrRNA with Cas9 mRNA or Cas9 protein on the genome editing activity. We previously reported that the disruption of tyrosinase (tyr) by tyr-gRNA/Cas9 mRNA causes a retinal pigment defect, whereas the disruption of spns2 by spns2-gRNA1/Cas9 mRNA leads to a cardiac progenitor migration defect in zebrafish. Here, we found that the injection of spns2-crRNA1, tyr-crRNA and tracrRNA with Cas9 mRNA or Cas9 protein simultaneously caused a migration defect in cardiac progenitors and a pigment defect in retinal epithelial cells. A time course analysis demonstrated that the injection of crRNAs and tracrRNA with Cas9 protein rapidly induced genome modifications compared with the injection of crRNAs and tracrRNA with Cas9 mRNA. We further show that the crRNA-tracrRNA-Cas9 protein complex is functional for the visualization of endogenous gene expression; therefore, this is a very powerful, ready-to-use system in zebrafish.
authors.affiliation: Laboratory for Developmental Biology, Center for Medical Education and Sciences, Graduate School of Medical Science, University of Yamanashi, Shimokato 1110, Chuo, Yamanashi, Japan.
authors.forename: Hirohito
authors.initials: H
authors.lastname: Kotani
authors.affiliation: Laboratory for Developmental Biology, Center for Medical Education and Sciences, Graduate School of Medical Science, University of Yamanashi, Shimokato 1110, Chuo, Yamanashi, 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, Japan.
authors.forename: Rie
authors.initials: R
authors.lastname: Ohga
authors.affiliation: Laboratory for Developmental Biology, Center for Medical Education and Sciences, Graduate School of Medical Science, University of Yamanashi, Shimokato 1110, Chuo, Yamanashi, 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, Japan.
authors.forename: Atsuo
authors.initials: A
authors.lastname: Kawahara
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D064130:CRISPR-Associated Proteins / Q000235:genetics*; D064113:CRISPR-Cas Systems; D002352:Carrier Proteins / Q000235:genetics*; D053903:DNA Breaks, Double-Stranded; D016678:Genome; D008565:Membrane Proteins / Q000235:genetics*; D014442:Monophenol Monooxygenase / Q000235:genetics*; D040641:Quantitative Trait Loci; D000094704:RNA, Guide, CRISPR-Cas Systems; D015027:Zebrafish / Q000235:genetics*; D029961:Zebrafish Proteins / Q000235:genetics*
chemical_list: D064130:CRISPR-Associated Proteins; D002352:Carrier Proteins; D008565:Membrane Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D029961:Zebrafish Proteins; C534950:sphingolipid transporter 2, zebrafish; D014442:Monophenol Monooxygenase