204 Blood Analysis of Cas9-Expressing Cattle

Abstract

The CRISPR/Cas9 system has proved to be a powerful tool for knockout and knock-in in various species. When 2 components—Cas9 and single guide (sg)RNA—are delivered into cells or embryos, the events of gene editing occur. Because Cas9 is essential for gene editing in the CRISPR/Cas9 system, some studies have reported the production of Cas9-expressing animals, such as mice, which could be used to increase gene editing efficiency in subsequent experiments. In previous reports, we successfully produced 4 Cas9-expressing cattle via microinjection (Hahn et al. 2016 Reprod. Fertil. Dev. 29, 211). Primary cells from these calves had Cas9 activity because transfection of only sgRNA resulted in gene deletion. The aim of this study was to analyse the blood of the transgenic cattle to investigate the effect of Cas9 expression on health. Two of 4 transgenic calves died; one had severe ruminant tympany, failed to respond to treatment, and died at 4 months of age, and the other died at 5 months of age due to accidental ingestion of a needle from a feed bunk. Blood samples were obtained from the surviving 2 transgenic cattle (1 male and 1 female) at 7 and 12 months for blood analysis. Five milliliters of whole blood samples was collected from the jugular vein. Portions were used for CBC (Hemavet 950, Drew Scientific, Miami Lakes, FL, USA) and for serum chemistry analysis (BS-400, Mindray, Shenzhen, China). Average values for white blood cells (9600 and 1057/mm³), neutrophils (4590 and 3870/mm³), lymphocytes (4020 and 5910/mm³), red blood cells (732,000 and 798,000/mm³), hemoglobin (9.5 and 10.2 g dL⁻¹), packed cell volume (24.3 and 25.3%), platelet (439,000 and 327,500/mm³), AST (76 and 104 IU), ALP (140 and 133 IU), BUN (7.5 and 10.5 mg dL⁻¹), and creatinine (1.3 and 1.0 mg dL⁻¹) of male and female transgenic calves were within the reference range. Additionally, there was no difference in general health information, including body temperature and feeding. In conclusion, we demonstrated that continuous Cas9 expression in transgenic cattle did not affect health status of the surviving calves in terms of blood analysis. They have grown up without any health issues and are currently 14 (female) and 15 (male) months old. In the near future, we will evaluate their germline transmission by natural breeding or in vitro fertilization.

This work was supported by BK21 PLUS Program for Creative Veterinary Science, NRF (NRF-2017R1A2B3004972), and Seoul Milk Coop (SNU 550–20160004).