95 PRODUCTION OF GENE-EDITED PIGS, CATTLE, AND LAMBS BY EMBRYO INJECTION OF TALENS OR ZFNs

Abstract

Transcription activator-like effector nuclease (TALEN) and zinc finger nuclease (ZFN) DNA editing technology enables site-directed engineering of the genome. To date, all gene-edited large animals have been produced by treatment of somatic cells and cloning to produce gene-edited offspring. Although effective, it does not take advantage of the ‘leave-no-trace' aspect of site-specific nucleases, and the derivation of food animals by cloning is negatively perceived by the public. Thus, we have investigated production of gene-edited pigs, cattle and sheep by direct injection of TALEN or ZFN mRNAs to develop loss-of-function alleles for disease resistance (RELA) or enhanced meat production (GDF8). In vitro studies demonstrated activity of TALENs by cytoplasmic injection of mRNAs from dosages of 2 to 20 ng mL–1 with an apparent increase in both editing frequency and toxicity at high dosage. Our first pregnancies were produced by transfer of pig embryos (in vivo produced) injected with 2 ng mL–1 RELA TALEN mRNA. Pregnancy was confirmed in 5 of 7 recipients 4 of which went full term giving rise to 39 piglets, 8 of which carried editing events (21%). In parallel, we injected ZFN mRNA (2 ng mL–1) targeted to a similar site of the RELA gene and 2 of 2 recipients became pregnant, resulting in the birth of 9 piglets, one of which was edited (11%). For cattle injections, we derived zygotes by ovum pickup from selected Nelore dams followed by in vitro maturation and fertilization with Nelore semen. Low (2 ng mL–1) and medium (5 ng mL–1) dosages of GDF8-targeted TALENs resulted in Day 7 development to morula/blastocyst stage in 40% (n = 18) and 10% (n = 66) of cultured embryos, respectively. A total of 20 morula/blastocysts were chosen for transfer to 11 recipients, resulting in two full-term pregnancies. One pregnancy produced two calves, both of which carried edited GDF8 alleles. Complications with parturition of the second pregnancy resulted in 2 stillborn calves, the genotypes of which are under investigation. Finally, 2 ng mL–1 of TALEN mRNA targeted to ovine GDF8 was injected into in vivo-produced sheep zygotes and transferred into nine recipients, 3 blastocysts each. The pregnancy rate, number of live-born animals, and gene editing frequency is under investigation and will be reported.