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Location: Animal Biosciences & Biotechnology Laboratory

Title: Targeted gene knock-in by CRISPR/Cas ribonucleoproteins in porcine zygotes

item PARK, KI-EUN - University Of Maryland
item Powell, Anne
item SANDMAIER, SHELLEY - University Of Maryland
item KIM, CHAN-MI - University Of Maryland
item MILEHAM, ALAN - Genus
item Donovan, David
item TELUGU, BHANU - University Of Maryland

Submitted to: Stem Cells and Development
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/14/2017
Publication Date: 2/14/2017
Citation: Park, K., Powell, A.M., Sandmaier, S.E., Kim, C., Mileham, A., Donovan, D.M., Telugu, B.P. 2017. Targeted gene knock-in by CRISPR/Cas ribonucleoproteins in porcine zygotes. Stem Cells and Development. doi: 10.1038/srep42458.

Interpretive Summary: There is a need for less expensive tools [than cloning], for the creation of genetically modified livestock. This work describes the use of genome editing tools applied directly to the newly formed embryo with the end result that the genetically modified livestock can be created in a matter of weeks rather than months or years. This discovery has the potential to be highly efficient and well received by the public thus allowing genome edited livestock to be available for use in both biomedical research and for human consumption. The nature of the genetic modifications are varied, but include both genetic changes that are indistinguishable from naturally occurring mutations as well as those changes in DNAthat are modified in comparison to wild types. This finding is expected to benefit both farmers and consumers and revolutionize the means by which genetically modified livestock can be created.

Technical 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 that even though mouse is a powerhouse genetic model, there is a requirement for large animal models such as pigs that can either serve as an alternative, or complement investigations from the mouse. However, the use of pig or other domesticated animal has not proven popular to date due in part to the technical difficulties and time required in generating animal models with desired genetic modifications. In this regard, the ability to directly modify the genome in the zygote and generate edited animals from those embryos is highly desirable. In this report, we have shown for the first time, the targeted gene knock-ins of short stretch of DNA sequences into porcine embryos by direct injections of Cas9 ribonucleoprotein complex and the generation of gene targeted animals. The Cas9 protein from Streptococcus pyogenes was pre-complexed with a single guide RNA targeting downstream of the ubiquitously expressed COL1A locus, and the complex alongside a single-stranded targeting oligo was microinjected into 1- cell porcine embryos. Using this approach a line of pigs has been generated that carry pseudo attP sites within the COL1A locus to enable phiC31 integrase mediated introduction of transgenes. In addition, we have also demonstrated the feasibility of knocking-in long transgenes as well. This new route for genome engineering in pigs through targeted knock-ins via zygote injection should greatly enhance applications in both agriculture and biomedicine.