title: Targeted gene knock-in by CRISPR/Cas ribonucleoproteins in porcine zygotes.
abstract: The domestic pig is an important "dual purpose" animal model for agricultural and biomedical applications. There is an emerging consensus in the biomedical community for the use of large animal models such as pigs to either serve as an alternative, or complement investigations from the mouse. However, the use of pig has not proven popular due to technical difficulties and time required in generating models with desired genetic modifications. In this regard, the ability to directly modify the genome in the zygote and generate edited animals is highly desirable. This report demonstrates for the first time, the generation of gene targeted animals by direct injection of Cas9 ribonucleoprotein complex and short stretches of DNA sequences into porcine zygotes. The Cas9 protein from Streptococcus pyogenes was pre-complexed with a single guide RNA targeting downstream of the ubiquitously expressed COL1A gene, and co-injected with a single-stranded repair template into porcine zygotes. Using this approach a line of pigs that carry pseudo attP sites within the COL1A locus to enable phiC31 integrase mediated introduction of transgenes has been generated. This new route for genome engineering in pigs via zygote injection should greatly enhance applications in both agriculture and biomedicine.
authors.affiliation: Department of Animal and Avian Sciences, University of Maryland, MD, USA.|Animal Bioscience and Biotechnology Laboratory, USDA-ARS, Beltsville, MD, USA.|Renovate Biosciences Inc, Reisterstown, MD, USA.
authors.forename: Ki-Eun
authors.initials: KE
authors.lastname: Park
authors.affiliation: Animal Bioscience and Biotechnology Laboratory, USDA-ARS, Beltsville, MD, USA.
authors.forename: Anne
authors.initials: A
authors.lastname: Powell
authors.affiliation: Department of Animal and Avian Sciences, University of Maryland, MD, USA.|Animal Bioscience and Biotechnology Laboratory, USDA-ARS, Beltsville, MD, USA.
authors.forename: Shelley E S
authors.initials: SE
authors.lastname: Sandmaier
authors.affiliation: Department of Animal and Avian Sciences, University of Maryland, MD, USA.|Animal Bioscience and Biotechnology Laboratory, USDA-ARS, Beltsville, MD, USA.
authors.forename: Chan-Mi
authors.initials: CM
authors.lastname: Kim
authors.affiliation: Genus plc, DeForest, WI, USA.
authors.forename: Alan
authors.initials: A
authors.lastname: Mileham
authors.affiliation: Animal Bioscience and Biotechnology Laboratory, USDA-ARS, Beltsville, MD, USA.
authors.forename: David M
authors.initials: DM
authors.lastname: Donovan
authors.affiliation: Department of Animal and Avian Sciences, University of Maryland, MD, USA.|Animal Bioscience and Biotechnology Laboratory, USDA-ARS, Beltsville, MD, USA.|Renovate Biosciences Inc, Reisterstown, MD, USA.
authors.forename: Bhanu P
authors.initials: BP
authors.lastname: Telugu
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D064113:CRISPR-Cas Systems; D024042:Collagen Type I / Q000235:genetics; D055879:Gene Knock-In Techniques; D055786:Gene Knockout Techniques; D023061:Gene Order; D018390:Gene Targeting; D056426:Genetic Loci; D005822:Genetic Vectors / Q000235:genetics; D059765:Homologous Recombination; D046912:Multiprotein Complexes; D011485:Protein Binding; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics; D012261:Ribonucleoproteins / Q000235:genetics*; D013552:Swine; D015053:Zygote / Q000378:metabolism*
chemical_list: D024042:Collagen Type I; D046912:Multiprotein Complexes; D012097:Repressor Proteins; D012261:Ribonucleoproteins