Somatic cell nuclear transfer followed by CRIPSR/CAS9 microinjection results in highly efficient genome editing in cloned pigs

Authors: SHEETS, TIMOTHY - US Department Of Agriculture (USDA); PARK, CHI-HUN - US Department Of Agriculture (USDA); PARK, KI-EUN - US Department Of Agriculture (USDA); Powell, Anne; Donovan, David; TELUGU, BHANU - University Of Maryland

Submitted to: International Journal of Molecular Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/23/2016
Publication Date: 9/14/2016
Citation: Sheets, T.P., Park, C., Park, K., Powell, A.M., Donovan, D.M., Telugu, B.P. 2016. Somatic cell nuclear transfer followed by CRIPSR/CAS9 microinjection results in highly efficient genome editing in cloned pigs. International Journal of Molecular Sciences. doi: 10.3390/ijms17122031.

Interpretive Summary: There is a need for non-rodent animal models for both biomedical and agricultural research. The pig is an ideal model for both human and veterinary applications. One of the primary draw-backs is the long generational time for pigs vs. rodents (years vs. weeks). In the rodent most genome modification approaches produced genetic change(s) in one copy of the gene and then the rodent is bred to homozygosity to create an animal with both gene copies (alleles) changed. The long generational time for the pig would make this unacceptable. This work reports on biallelic (both gene copies changed) after cytoplasmic injections into the single cell pig embryo with genome editing enzymes (CRISPR/Cas9 complexes), such that the first pigs produced (founders) are homozygous for the genetic change of interest (thus avoiding the breeding to homozygosity). Similarly, in order to obtain embryos of a specific genetic background, a large founder herd is usually required, along with timed ovulations/breeding to obtain single cell embryos. This requires a large resource investment to maintain a herd of the desired genetic background and the isolation of the single cell embryos. The work reported in this manuscript avoids this resource demand. It describes producing an Ossabaw embryo via somatic cell nuclear transfer (cloning, like the sheep Dolly), using an Ossabaw pig fetal fibroblast cell line as a nuclear donor that was transferred into commercially purchased oocytes [that subsequently had their nuclei removed]. This cloned embryo was then injected with genome editing enzymes allowing the creation of a cloned and genome edited Ossabaw pig, while avoiding the need for maintaining an Ossabaw herd. The impact of this work will be felt primarily by the genome editing scientific community and demonstrates the ability to create genome edited specialty pig breeds without the need for maintaining a specialty herd.

Technical Abstract: The domestic pig is an ideal “dual purpose” animal model for agricultural and biomedical research. With the availability of genome editing tools [e.g. clustered regularly interspersed 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 targeting GRB10 into the reconstituted oocytes to generate GRB10 ablated Ossabaw fetuses. This strategy resulted in highly efficient and effective 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 colony 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.