title: Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis.
abstract: We have assessed the efficacy of the recently developed CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system for genome modification in the amphibian Xenopus tropicalis. As a model experiment, targeted mutations of the tyrosinase gene were verified, showing the expected albinism phenotype in injected embryos. We further tested this technology by interrupting the six3 gene, which is required for proper eye and brain formation. Expected eye and brain phenotypes were observed when inducing mutations in the six3 coding regions, as well as when deleting the gene promoter by dual targeting. We describe here a standardized protocol for genome editing using this system. This simple and fast method to edit the genome provides a powerful new reverse genetics tool for Xenopus researchers.
authors.forename: Takuya
authors.initials: T
authors.lastname: Nakayama
authors.forename: Margaret B
authors.initials: MB
authors.lastname: Fish
authors.forename: Marilyn
authors.initials: M
authors.lastname: Fisher
authors.forename: Jamina
authors.initials: J
authors.lastname: Oomen-Hajagos
authors.forename: Gerald H
authors.initials: GH
authors.lastname: Thomsen
authors.forename: Robert M
authors.initials: RM
authors.lastname: Grainger
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004625:Embryo, Nonmammalian / Q000378:metabolism; D005123:Eye / Q000378:metabolism; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D056426:Genetic Loci; D016678:Genome; D005854:Germ Cells / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D014981:Xenopus / Q000196:embryology* / Q000235:genetics*; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism; D000097607:Homeobox Protein SIX3
chemical_list: D005136:Eye Proteins; D018398:Homeodomain Proteins; D009419:Nerve Tissue Proteins; D029867:Xenopus Proteins; D000097607:Homeobox Protein SIX3