136 Effects of the homology direct repair enhancer RS-1 on pig embryo culture

Advances in genome editing technologies, such as the CRISPR/Cas9 system, have facilitated gene manipulation and the generation of pigs resistant to disease, models for studying human diseases and for xenotransplantation. However, double-strand breaks generated by the CRISPR/Cas9 system are preferentially repaired by the non-homologous end joining (NHEJ) pathway compared with high-fidelity homology-directed repair (HDR). All reports of pigs created by zygote injection of the CRISPR/Cas9 system result from NHEJ rather than HDR. The molecule known as RS-1 was found to stimulate RAD51 and thus enhance HDR. Thus, our goals are to understand this pathway by evaluating the response, as well as dosage and temporal effects, of RS-1 on porcine embryo development, and to determine a safe concentration to achieve high HDR rates without affecting embryo development. As RS-1 was added to culture medium from a 7.5 mM stock solution in DMSO, we also evaluated whether RS-1 in solution could have affinity and migrate to the mineral oil overlay placed in the media during embryo culture. After IVF, embryos were cultured without RS-1 (control groups) or in the presence of RS-1 at 7.5 and 15 µM. At 7.5 µM, RS-1 improved HDR in rabbits created by zygote injection of the CRISPR/Cas9. Based on that, we designed five experimental groups to evaluate short- (A, 20 h) and long-term (B, 144 h) effects of RS-1: two groups in the presence of RS-1 at 7.5 µM (7.5 µM A and 7.5 µM B), two control groups (control A and control B), and a group in the presence of RS-1 at 15 µM A for short-term exposure only. First, embryos in the groups containing (7.5 µM A, 7.5 µM B, and 15 µM A) and without RS-1 (control A and control B) were cultured for 20 h without the mineral oil overlay. Then, embryos from control A, 7.5 µM A, and 15 µM A groups were washed and cultured without RS-1 until Day 6 in the presence of the mineral oil overlay. To evaluate long-term effects of RS-1, embryos from control B and 7.5 µM B groups were washed and transferred to medium without or with RS-1 at 7.5 µM, respectively, and cultured until Day 6 with no mineral oil overlay. We report the mean values for each set of data ± s.e.m., and the degree of statistical significance in all analyses was defined at P < 0.05. We observed that the presence of RS-1 at 15 µM decreased cleavage rates (control A 79.1 ± 2.7%; 7.5 µM A 83.6 ± 1.6%; 15 µM 70.9 ± 4.4%; control B 80.8 ± 1.4%; 7.5 µM B 80.0 ± 2.8%), and long-term exposure to RS-1 decreased development to the blastocyst stage (control A 46.5 ± 3.0%; 7.5 µM A 42.7 ± 3.4%; 15 µM 41.8 ± 4.7%; control B 44.9 ± 3.3%; 7.5 µM B 28.8 ± 4.9%). In addition, RS-1 exposure decreased the total cell number compared with the controls (control A 61.4 ± 2.8; 7.5 µM A 49.2 ± 3.0; 15 µM 48.5 ± 2.3; control B 59.9 ± 3.4; 7.5 µM B 41.5 ± 2.5), however no differences in apoptosis rates were observed between the treatments. Our work will serve as a basis to understand the effects of RS-1 and RAD51 during embryonic development and to improve the applicability of customizable nucleases for the production of genetically modified pigs.