13 Generation of SLICK beef cattle by embryo microinjection: A case report

Abstract

Due to climate change, cattle can experience heat stress more frequently in traditionally temperate, non-tropical environments. Because of this and the fact that most of the world’s inefficient cattle reside in tropical zones, we set out to demonstrate our ability to adapt Angus animals to heat stress through a single gene edit in the prolactin receptor (PRLR). We selected PRLR, because it is known that the SLICK phenotype results from 1 of 3 different mutations in PRLR and therefore is related to heat stress regulation. The overall project was initiated through a partnership between Acceligen and BluePrint Genetics (BPG). BPG harvested 1415 oocytes from a total of 9 ovum pickup rounds from 7 different Angus donors. These oocytes were graded, selected, and put in maturation medium for shipping to Acceligen. Matured oocytes were fertilized in Fert-TALP medium and co-incubated with frozen/thawed semen from 3 different reproductive certified Angus bulls. Consequently, targeted editing was done on a single cell in the zygotes 12 h after fertilization by introduction of guide (g)RNA/Cas9 (250 ng μL⁻¹ of each) through intracytoplasmic microinjection. Then, treated zygotes (n = 1341) were cultured in synthetic oviductal fluid with amino acids (SOFaa) culture medium under a controlled atmosphere until on Day 6, when 277 (20%) grade 1 embryos were selected and 39 returned to Blueprint Genetics for transfer. Because the number of available recipients was limited, only 4 rounds of fresh embryo transfer were completed, and the remaining embryos (n = 190) were vitrified for future transfers. Pregnancy checks by ultrasound on Day 30 revealed a 23% (9/39) rate of pregnancy, which decreased to 13% (5/39) by Day 60. In total, 4 animals reached term and delivered healthy calves. Genetic testing of the PRLR target site was done by amplicon sequencing, which showed 3 edited SLICK animals (75%) and one wild-type. Thus, the SLICK zygote editing by microinjection was demonstrated to be an efficient method to produce bovine beef animals and is currently being prepared for regulatory review in multiple countries and commercialization.