Targeted Gene Knockin in Porcine Somatic Cells Using CRISPR/Cas Ribonucleoproteins

Ki-Eun Park; Chi-Hun Park; Anne Powell; Jessica Martin; David M Donovan; Bhanu P Telugu

doi:10.3390/ijms17060810

Targeted Gene Knockin in Porcine Somatic Cells Using CRISPR/Cas Ribonucleoproteins

Int J Mol Sci. 2016 May 26;17(6):810. doi: 10.3390/ijms17060810.

Authors

Ki-Eun Park^{1

2

3}, Chi-Hun Park^{4

5}, Anne Powell⁶, Jessica Martin^{7

8}, David M Donovan⁹, Bhanu P Telugu^{10

11

12}

Affiliations

¹ Department of Animal and Avian Sciences, University of Maryland, College Park, MD 20742, USA. kepark@umd.edu.
² Animal Bioscience and Biotechnology Laboratory, USDA-ARS, Beltsville, MD 20705, USA. kepark@umd.edu.
³ Livestock Genomix, Reisterstown, MD 21136, USA. kepark@umd.edu.
⁴ Department of Animal and Avian Sciences, University of Maryland, College Park, MD 20742, USA. chpark@umd.edu.
⁵ Animal Bioscience and Biotechnology Laboratory, USDA-ARS, Beltsville, MD 20705, USA. chpark@umd.edu.
⁶ Animal Bioscience and Biotechnology Laboratory, USDA-ARS, Beltsville, MD 20705, USA. anne.powell@ars.usda.gov.
⁷ Department of Animal and Avian Sciences, University of Maryland, College Park, MD 20742, USA. jjm11393@umd.edu.
⁸ Animal Bioscience and Biotechnology Laboratory, USDA-ARS, Beltsville, MD 20705, USA. jjm11393@umd.edu.
⁹ Animal Bioscience and Biotechnology Laboratory, USDA-ARS, Beltsville, MD 20705, USA. david.donovan@ars.usda.gov.
¹⁰ Department of Animal and Avian Sciences, University of Maryland, College Park, MD 20742, USA. btelugu@umd.edu.
¹¹ Animal Bioscience and Biotechnology Laboratory, USDA-ARS, Beltsville, MD 20705, USA. btelugu@umd.edu.
¹² Livestock Genomix, Reisterstown, MD 21136, USA. btelugu@umd.edu.

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.

Keywords: CRISPR/Cas; SCNT; TARGATT pig; cloning; knockin; transegenic.

MeSH terms

Animals
Animals, Genetically Modified
Attachment Sites, Microbiological
CRISPR-Cas Systems
Cells, Cultured
Collagen Type I / genetics*
Fibroblasts / cytology
Gene Knock-In Techniques / methods*
Gene Targeting
Genetic Engineering / methods
Humans
Integrases / metabolism
Nuclear Transfer Techniques
Ribonucleoproteins / metabolism*
Swine / genetics*

Substances

Collagen Type I
Ribonucleoproteins
Integrases