title: Precise genome editing by homologous recombination.
abstract: Simple and efficient methods are presented for creating precise modifications of the zebrafish genome. Edited alleles are generated by homologous recombination between the host genome and double-stranded DNA (dsDNA) donor molecules, stimulated by the induction of double-strand breaks at targeted loci in the host genome. Because several kilobase-long tracts of sequence can be exchanged, multiple genome modifications can be generated simultaneously at a single locus. Methods are described for creating: (1) alleles with simple sequence changes or in-frame additions, (2) knockin/knockout alleles that express a reporter protein from an endogenous locus, and (3) conditional alleles in which exons are flanked by recombinogenic loxP sites. Significantly, our approach to genome editing allows the incorporation of a linked reporter gene into the donor sequences so that successfully edited alleles can be identified by virtue of expression of the reporter. Factors affecting the efficiency of genome editing are discussed, including the finding that dsDNA products of I-SceI meganuclease enzyme digestion are particularly effective as donor molecules for gene-editing events. Reagents and procedures are described for accomplishing efficient genome editing in the zebrafish.
authors.affiliation: University of Utah, Salt Lake City, UT, United States.
authors.forename: K
authors.initials: K
authors.lastname: Hoshijima
authors.affiliation: University of Utah, Salt Lake City, UT, United States.
authors.forename: M J
authors.initials: MJ
authors.lastname: Jurynec
authors.affiliation: University of Utah, Salt Lake City, UT, United States.
authors.forename: D J
authors.initials: DJ
authors.lastname: Grunwald
mesh_terms: D000818:Animals; D004247:DNA / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics; D000072669:Gene Editing / Q000379:methods*; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D016678:Genome / Q000235:genetics; D059765:Homologous Recombination / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D015027:Zebrafish / Q000235:genetics
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; C066166:AI4 protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific
keywords: Conditional mutation; Genome editing; Homologous recombination; Knockin mutation; Zebrafish