title: Precise and efficient genome editing in zebrafish using the CRISPR/Cas9 system.
abstract: The introduction of engineered site-specific DNA endonucleases has brought precise genome editing in many model organisms and human cells into the realm of possibility. In zebrafish, loss-of-function alleles have been successfully produced; however, germ line transmission of functional targeted knock-ins of protein tags or of SNP exchanges have not been reported. Here we show by phenotypic rescue that the CRISPR/Cas system can be used to target and repair a premature stop codon at the albino (alb) locus in zebrafish with high efficiency and precision. Using circular donor DNA containing CRISPR target sites we obtain close to 50% of larvae with precise homology-directed repair of the alb(b4) mutation, a small fraction of which transmitted the repaired allele in the germ line to the next generation (3/28 adult fish). The in vivo demonstration of germ line transmission of a precise SNP exchange in zebrafish underscores its suitability as a model for genetic research.
authors.affiliation: Max-Planck-Institut für Entwicklungsbiologie, Spemannstr. 35, Tübingen 72076, Germany uwe.irion@tuebingen.mpg.de.
authors.forename: Uwe
authors.initials: U
authors.lastname: Irion
authors.affiliation: Max-Planck-Institut für Entwicklungsbiologie, Spemannstr. 35, Tübingen 72076, Germany.
authors.forename: Jana
authors.initials: J
authors.lastname: Krauss
authors.affiliation: Max-Planck-Institut für Entwicklungsbiologie, Spemannstr. 35, Tübingen 72076, Germany.
authors.forename: Christiane
authors.initials: C
authors.lastname: Nüsslein-Volhard
mesh_terms: D000818:Animals; D064130:CRISPR-Associated Proteins / Q000235:genetics; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D018389:Codon, Nonsense / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004260:DNA Repair / Q000235:genetics*; D004270:DNA, Circular / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D016678:Genome / Q000235:genetics*; D005838:Genotype; D026901:Membrane Transport Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D015027:Zebrafish / Q000235:genetics*; D029961:Zebrafish Proteins / Q000235:genetics*
chemical_list: D064130:CRISPR-Associated Proteins; D018389:Codon, Nonsense; D017931:DNA Primers; D004270:DNA, Circular; D026901:Membrane Transport Proteins; C583611:Slc45a2 protein, zebrafish; D029961:Zebrafish Proteins
keywords: CRISPR/Cas; Genome editing; Zebrafish; albino; slc45a2