title: Efficient introgression of allelic variants by embryo-mediated editing of the bovine genome.
abstract: The recent development of designer nucleases allows for the efficient and precise introduction of genetic change into livestock genomes. Most studies so far have focused on the introduction of random mutations in cultured cells and the use of nuclear transfer to generate animals with edited genotypes. To circumvent the intrinsic uncertainties of random mutations and the inefficiencies of nuclear transfer we directed our efforts to the introduction of specific genetic changes by homology-driven repair directly in in vitro produced embryos. Initially, we injected zinc finger nuclease (ZFN)-encoding mRNA or DNA into bovine zygotes to verify cleavage activity at their target site within the gene for beta-lactoglobulin (LGB) and detected ZFN-induced random mutations in 30% to 80% of embryos. Next, to precisely change the LGB sequence, we co-injected ZFNs or transcription activator-like effector nucleases (TALENs) with DNA oligonucleotides (ODNs). Analysis of co-injected embryos showed targeted changes in up to 33% (ZFNs) and 46% (TALENs) of blastocysts. Deep sequence analysis of selected embryos revealed contributions of the targeted LGB allele can reach 100% which implies that genome editing by zygote injections can facilitate the one-step generation of non-mosaic livestock animals with pre-designed biallelic modifications.
authors.affiliation: 1] AgResearch, Ruakura, Hamilton, New Zealand [2] Guangxi University, Nabbing, China.
authors.forename: Jingwei
authors.initials: J
authors.lastname: Wei
authors.affiliation: AgResearch, Ruakura, Hamilton, New Zealand.
authors.forename: Stefan
authors.initials: S
authors.lastname: Wagner
authors.affiliation: 1] AgResearch, Ruakura, Hamilton, New Zealand [2] China Agricultural University, Beijing, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Lu
authors.affiliation: AgResearch, Ruakura, Hamilton, New Zealand.
authors.forename: Paul
authors.initials: P
authors.lastname: Maclean
authors.affiliation: Recombinetics, St. Paul, Minnesota, USA.
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Carlson
authors.affiliation: Recombinetics, St. Paul, Minnesota, USA.
authors.forename: Scott C
authors.initials: SC
authors.lastname: Fahrenkrug
authors.affiliation: AgResearch, Ruakura, Hamilton, New Zealand.
authors.forename: Götz
authors.initials: G
authors.lastname: Laible
mesh_terms: D000483:Alleles; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004720:Endonucleases / Q000378:metabolism; D005818:Genetic Engineering; D056426:Genetic Loci; D014644:Genetic Variation; D016678:Genome; D054643:INDEL Mutation; D007782:Lactoglobulins / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D053652:Nuclear Transfer Techniques; D016415:Sequence Alignment; D016335:Zinc Fingers
chemical_list: D007782:Lactoglobulins; D004720:Endonucleases