title: Somatic Cell Nuclear Transfer Followed by CRIPSR/Cas9 Microinjection Results in Highly Efficient Genome Editing in Cloned Pigs.
abstract: The domestic pig is an ideal "dual purpose" animal model for agricultural and biomedical research. With the availability of genome editing tools such as clustered regularly interspaced short palindromic repeat (CRISPR) and associated nuclease Cas9 (CRISPR/Cas9), it is now possible to perform site-specific alterations with relative ease, and will likely help realize the potential of this valuable model. In this article, we investigated for the first time a combination of somatic cell nuclear transfer (SCNT) and direct injection of CRISPR/Cas ribonucleoprotein complex targetingGRB10into the reconstituted oocytes to generateGRB10ablated Ossabaw fetuses. This strategy resulted in highly efficient (100%) generation of biallelic modifications in cloned fetuses. By combining SCNT with CRISPR/Cas9 microinjection, genome edited animals can now be produced without the need to manage a founder herd, while simultaneously eliminating the need for laborious in vitro culture and screening. Our approach utilizes standard cloning techniques while simultaneously performing genome editing in the cloned zygotes of a large animal model for agriculture and biomedical applications.
authors.affiliation: Department of Animal and Avian Sciences, University of Maryland, College Park, MD 20742, USA. tsheets@umd.edu.|Animal Bioscience and Biotechnology Laboratory, USDA-ARS, Beltsville, MD 20705, USA. tsheets@umd.edu.
authors.forename: Timothy P
authors.initials: TP
authors.lastname: Sheets
authors.affiliation: Department of Animal and Avian Sciences, University of Maryland, College Park, MD 20742, USA. chpark@umd.edu.|Animal Bioscience and Biotechnology Laboratory, USDA-ARS, Beltsville, MD 20705, USA. chpark@umd.edu.
authors.forename: Chi-Hun
authors.initials: CH
authors.lastname: Park
authors.affiliation: Department of Animal and Avian Sciences, University of Maryland, College Park, MD 20742, USA. kepark@umd.edu.|Animal Bioscience and Biotechnology Laboratory, USDA-ARS, Beltsville, MD 20705, USA. kepark@umd.edu.|Renovate Biosciences Inc., Reisterstown, MD 21136, USA. kepark@umd.edu.
authors.forename: Ki-Eun
authors.initials: KE
authors.lastname: Park
authors.affiliation: Animal Bioscience and Biotechnology Laboratory, USDA-ARS, Beltsville, MD 20705, USA. anne.powell@ars.usda.gov.
authors.forename: Anne
authors.initials: A
authors.lastname: Powell
authors.affiliation: Animal Bioscience and Biotechnology Laboratory, USDA-ARS, Beltsville, MD 20705, USA. david.donovan@ars.usda.gov.
authors.forename: David M
authors.initials: DM
authors.lastname: Donovan
authors.affiliation: Department of Animal and Avian Sciences, University of Maryland, College Park, MD 20742, USA. btelugu@umd.edu.|Animal Bioscience and Biotechnology Laboratory, USDA-ARS, Beltsville, MD 20705, USA. btelugu@umd.edu.|Renovate Biosciences Inc., Reisterstown, MD 21136, USA. btelugu@umd.edu.
authors.forename: Bhanu P
authors.initials: BP
authors.lastname: Telugu
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D019976:Cloning, Organism; D000072669:Gene Editing / Q000379:methods*; D016678:Genome; D060005:Genotyping Techniques; D008845:Microinjections / Q000379:methods*; D053652:Nuclear Transfer Techniques; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000378:metabolism; D034421:Sus scrofa / Q000235:genetics*; D015053:Zygote / Q000378:metabolism
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems
keywords: CRISPR/Cas; SCNT; knockout; microinjection; pig