title: Targeted Gene Knockin in Porcine Somatic Cells Using CRISPR/Cas Ribonucleoproteins.
abstract: The pig is an ideal large animal model for genetic engineering applications. A relatively short gestation interval and large litter size makes the pig a conducive model for generating and propagating genetic modifications. The domestic pig also shares close similarity in anatomy, physiology, size, and life expectancy, making it an ideal animal for modeling human diseases. Often, however, the technical difficulties in generating desired genetic modifications such as targeted knockin of short stretches of sequences or transgenes have impeded progress in this field. In this study, we have investigated and compared the relative efficiency of CRISPR/Cas ribonucleoproteins in engineering targeted knockin of pseudo attP sites downstream of a ubiquitously expressed COL1A gene in porcine somatic cells and generated live fetuses by somatic cell nuclear transfer (SCNT). By leveraging these knockin pseudo attP sites, we have demonstrated subsequent phiC31 integrase mediated integration of green fluorescent protein (GFP) transgene into the site. This work for the first time created an optimized protocol for CRISPR/Cas mediated knockin in porcine somatic cells, while simultaneously creating a stable platform for future transgene integration and generating transgenic animals.
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.|Livestock Genomix, Reisterstown, MD 21136, USA. kepark@umd.edu.
authors.forename: Ki-Eun
authors.initials: KE
authors.lastname: Park
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: 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: Department of Animal and Avian Sciences, University of Maryland, College Park, MD 20742, USA. jjm11393@umd.edu.|Animal Bioscience and Biotechnology Laboratory, USDA-ARS, Beltsville, MD 20705, USA. jjm11393@umd.edu.
authors.forename: Jessica
authors.initials: J
authors.lastname: Martin
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.|Livestock Genomix, Reisterstown, MD 21136, USA. btelugu@umd.edu.
authors.forename: Bhanu P
authors.initials: BP
authors.lastname: Telugu
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001287:Attachment Sites, Microbiological; D064113:CRISPR-Cas Systems; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics*; D005347:Fibroblasts / Q000166:cytology; D055879:Gene Knock-In Techniques / Q000379:methods*; D018390:Gene Targeting; D005818:Genetic Engineering / Q000379:methods; D006801:Humans; D019426:Integrases / Q000378:metabolism; D053652:Nuclear Transfer Techniques; D012261:Ribonucleoproteins / Q000378:metabolism*; D013552:Swine / Q000235:genetics*
chemical_list: D024042:Collagen Type I; D012261:Ribonucleoproteins; D019426:Integrases
keywords: CRISPR/Cas; SCNT; TARGATT pig; cloning; knockin; transegenic