title: CRISPR/Cas9-mediated gene knockout in the ascidian Ciona intestinalis.
abstract: Knockout of genes with CRISPR/Cas9 is a newly emerged approach to investigate functions of genes in various organisms. We demonstrate that CRISPR/Cas9 can mutate endogenous genes of the ascidian Ciona intestinalis, a splendid model for elucidating molecular mechanisms for constructing the chordate body plan. Short guide RNA (sgRNA) and Cas9 mRNA, when they are expressed in Ciona embryos by means of microinjection or electroporation of their expression vectors, introduced mutations in the target genes. The specificity of target choice by sgRNA is relatively high compared to the reports from some other organisms, and a single nucleotide mutation at the sgRNA dramatically reduced mutation efficiency at the on-target site. CRISPR/Cas9-mediated mutagenesis will be a powerful method to study gene functions in Ciona along with another genome editing approach using TALE nucleases.
authors.affiliation: Shimoda Marine Research Center, University of Tsukuba, Shimoda, Shizuoka, 415-0025, Japan.
authors.forename: Haruka
authors.initials: H
authors.lastname: Sasaki
authors.forename: Keita
authors.initials: K
authors.lastname: Yoshida
authors.forename: Akiko
authors.initials: A
authors.lastname: Hozumi
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Sasakura
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics / Q000502:physiology*; D002938:Ciona intestinalis / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D018274:Electroporation; D055786:Gene Knockout Techniques / Q000379:methods*; D005801:Genes, Homeobox / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D023281:Genomics / Q000379:methods; D016296:Mutagenesis / Q000235:genetics*; D059645:Mutation Rate; D016133:Polymerase Chain Reaction
chemical_list: D017931:DNA Primers
keywords: CRISPR/Cas9; Ciona intestinalis; ascidian; knockout; mutagenesis