|PARK, KI-EUN - University Of Maryland|
|PARK, CHI-HUN - University Of Maryland|
|TELUGU, BHANU - University Of Maryland|
Submitted to: International Journal of Molecular Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/19/2016
Publication Date: 5/26/2016
Citation: Park, K., Park, C., Powell, A.M., Donovan, D.M., Telugu, B.P. 2016. Targeted gene knockin in porcine somatic cells using CRISPR/Cas ribonucleoproteins. International Journal of Molecular Sciences. 17(6).
Interpretive Summary: In light of the fact that transgenic and cloned animals have been approved by the FDA for human consumption, there is a need for easily manipulated livestock cell lines that are amenable to somatic cell nuclear transfer (SCNT; cloning) and which can receive transgenes at specific loci in the genome (to avoid the vagaries of randomly integrated transgenes). Pig is an ideal model for both biomedical research and putative livestock improvements for agricultural applications and human consumption. In order to create a pig cell line that is poised to receive transgenes at a specific site in the genome, genome editing enzyme complexes (CRISPR/Cas9) were used to introduce modified sites (AttP) at the COL1A gene in the pig genome. The AttP sites are known to integrate transgenes at a high frequency under certain conditions (in the presence of phiC32 integrase). As a proof of concept, an easily monitored reporter gene (green fluorescent protein; GFP) was introduced into this site and shown to express as expected. This strategy and cell line should help other scientists to create similar constructs in other livestock breeds to aid in the creation of genetically engineered livestock with controlled sites of integration.
Technical Abstract: The domestic 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 locus 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 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.