(270) Effect of Different Electroporation Voltages on the Embryo Viability and the Indel Efficiency of the CRISPR-RNP Complex Targeting the Beta-Lactoglobulin Gene in  In Vitro Generated Bovine Embryos

M. O. Atli¹ ,  T. Karaşahin², G. Şentürk², Ş. Dursun³, M. Kose⁴, M. Hitit⁵, Y. Keskinbıcak⁶,  S. Özdemir⁶, R. Sevgi⁷, A. Okuroglu⁷, C. Baloglu⁸, F. Bozkaya⁶

1.  Department of Reproduction, Faculty of Veterinary Medicine, Harran University, Sanliurfa, Turkey

2. Department of Phisiology, Faculty of Veterinary Medicine, Aksaray University, Aksaray,Turkey

3. Department of Obstetrics, Faculty of Veterinary Medicine, Aksaray University, Aksaray,Turkey

4. Department of Obstetrics, Faculty of Veterinary Medicine, Dicle University, Diyarbakır,Turkey

5. College of Agriculture, Food and Natural Resources, Cooperative Agricultural Research Center, Prairie View A&M University, Prairie View, TX, United States of America

6. Department of Genetics, Faculty of Veterinary Medicine, Harran University, Sanliurfa, Turkey

 7. Department of Genetics, Faculty of Veterinary Medicine, Atatürk University, Erzurum, Turkey

7. Department of Biotechnology, International Livestock Research and Training Center, Ankara, Turkey

8. Department of Genetics, Faculty of Agriculture, Kastamonu University, Kastamonu, Turkey

Abstract Text: Considering its less dexterous and less time-consuming characteristics, electroporation has emerged as a better method for delivering ribonucleoproteins into the intact zona pellucida of presumptive zygotes in livestock. Previous reports suggest that different electroporation voltages have different effects on the viability and indel efficiency of in-vitro produced embryos in different livestock species. The aim of this study was to investigate the effects of different electroporation voltages on the embryo viability and the indel efficiency of the CRISPR-Cas9 Ribo-nucleo-Protein (RNP) complex targeting the exon 1 of beta-lactoglobulin gene (BLG) in in vitro-generated bovine embryos. For this purpose, putative bovine zygotes (PBZ) were subjected to electroporation in the electroporation cuvette (0.1 cm) at different voltages 6 hours after fertilization in OPTİ-MEM solution [control (0 V, n=186 oocytes), group I (15 V, n=220 oocytes), group II (20 V, n=218 oocytes) or group III (30 V, n=236 oocytes) ]. Each experiment was repeated four times, and each voltage was fixed with 5 pulses of 3 ms in length with 100 ms between each pulse. Synthetically generated two single-guide (sg) RNAs targeting the first exon of BLG were incubated with Cas9 protein prior to the experiment. The cleavage rate on day 2 and blastocyst rates on day 7 of PBZ were recorded. The indel rate (mocasis and bialelic mutation) of the blastocysts for the BLG gene from the experiments was assessed by both gel electrophoresis and Sanger sequencing. Data was analyzed by ANOVA.  Among the groups, the cleavage and blastocyst rate was lower only in group III (30 V) (37.9±7.9% and 9.4±2.1%, respectively, P< 0.001). On the other hand, we observed a lower mosaic and biallelic rate (11.2±1.1% and 5.3±0.7% respectively) in group I (15V), compared to group II (20V) and group III (30V). In summary, although both 20 V and 30 V induced higher indel rates in the blastosists tested, embryonic viability was lower in the group III (30 V) group, suggesting that 20 V may be ideal for generating BLG knockout cattle (This study was supported by TUBİTAK 122O486).