title: Rapid and efficient analysis of gene function using CRISPR-Cas9 in Xenopus tropicalis founders.
abstract: Recent advances in genome editing using programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system, have facilitated reverse genetics in Xenopus tropicalis. To establish a practical workflow for analyzing genes of interest using CRISPR-Cas9, we examined various experimental procedures and conditions. We first compared the efficiency of gene disruption between Cas9 protein and mRNA injection by analyzing genotype and phenotype frequency, and toxicity. Injection of X. tropicalis embryos with Cas9 mRNA resulted in high gene-disrupting efficiency comparable with that produced by Cas9 protein injection. To exactly evaluate the somatic mutation rates of on-target sites, amplicon sequencing and restriction fragment length polymorphism analysis using a restriction enzyme or recombinant Cas9 were performed. Mutation rates of two target genes (slc45a2 and ltk) required for pigmentation were estimated to be over 90% by both methods in animals exhibiting severe phenotypes, suggesting that targeted somatic mutations were biallelically introduced in almost all somatic cells of founder animals. Using a heteroduplex mobility assay, we also showed that off-target mutations were induced at a low rate. Based on our results, we propose a CRISPR-Cas9-mediated gene disruption workflow for a rapid and efficient analysis of gene function using X. tropicalis founders.
authors.affiliation: Department of Mathematical and Life Sciences, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8526, Japan.
authors.forename: Mitsuki
authors.initials: M
authors.lastname: Shigeta
authors.affiliation: Department of Mathematical and Life Sciences, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8526, Japan.
authors.forename: Yuto
authors.initials: Y
authors.lastname: Sakane
authors.affiliation: Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka, Fukuoka, 820-8502, Japan.
authors.forename: Midori
authors.initials: M
authors.lastname: Iida
authors.affiliation: Department of Mathematical and Life Sciences, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8526, Japan.
authors.forename: Miyuki
authors.initials: M
authors.lastname: Suzuki
authors.affiliation: Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8526, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Kashiwagi
authors.affiliation: Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8526, Japan.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Kashiwagi
authors.affiliation: Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka, Fukuoka, 820-8502, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Fujii
authors.affiliation: Department of Mathematical and Life Sciences, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8526, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Department of Mathematical and Life Sciences, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8526, Japan.
authors.forename: Ken-Ichi T
authors.initials: KT
authors.lastname: Suzuki
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D004625:Embryo, Nonmammalian; D047108:Embryonic Development / Q000235:genetics; D000072669:Gene Editing; D018390:Gene Targeting; D005818:Genetic Engineering; D009154:Mutation; D010641:Phenotype; D012333:RNA, Messenger / Q000235:genetics / Q000494:pharmacology*; D014981:Xenopus / Q000235:genetics* / Q000254:growth & development
chemical_list: D012333:RNA, Messenger