159 Stable germline transmission of multiple gene-edited bulls for precision breeding

D. H. Kwon; K. H. Eom; G. M. Gim; B. J. Jeon; J. Y. Choi; D. J. Jung; D. H. Kim; J. K. Yi; J. J. Ha; J. H. Lee; S. R. Han; S. B. Lee; S. Y. Yum; W. W. Lee; G. Jang

doi:10.1071/RDv36n2Ab159

RESEARCH ARTICLE

159 Stable germline transmission of multiple gene-edited bulls for precision breeding

D. H. Kwon ^A , K. H. Eom ^A ^C , G. M. Gim ^A ^C , B. J. Jeon ^A , J. Y. Choi ^A , D. J. Jung ^B , D. H. Kim ^B , J. K. Yi ^B , J. J. Ha ^B , J. H. Lee ^C , S. R. Han ^C , S. B. Lee ^C , S. Y. Yum ^C , W. W. Lee ^C and G. Jang ^A ^D

+ Author Affiliations

- Author Affiliations

^A Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul, Korea

^B Gyeongsangbukdo Livestock Research Institute, Seoul, Korea

^C LARTBio Inc., Yoeongju, Korea

^D Comparative Medicine Disease Research Center, Seoul National University, Seoul, Korea

Reproduction, Fertility and Development 36(2) 233 https://doi.org/10.1071/RDv36n2Ab159

Since the development of gene editing techniques, numerous attempts have been made to graft the techniques onto the agriculture sector. Double gene knockout cattle (targeting site; myostatin (MSTN)/prion protein (PRNP) and MSTN/β-lactoglobulin (BLG)) were produced utilising electroporation employing CRISPR/Cas9 (Gim et al. 2023 J. Anim. Sci. Biotechnol. 14, 103). Our goal in this study was to evaluate bulls generated using electroporation as significant resources for rapidly propagating desirable traits by demonstrating transmission of mutated gene along germ cells. For the purpose, semen was collected from the founder (F0) bulls. Using the sperm, we performed IVF with wildtype oocytes from a slaughterhouse to evaluate delivery of the mutation and fertility of the founder germ cell. The presence of the mutation in resultant embryos was analysed by T7 endonuclease assay and compared with mutation rates identified in somatic cells in F0 bulls previously: 99.3% for MSTN and 13.7% for PRNP in case of MSTN-/PRNP- bulls, 50.3% for MSTN and 55.3% for BLG in case of MSTN-/BLG- cattle. Oocytes were aspirated from ovaries from a slaughterhouse. After 22–24 h of maturation, the oocytes were co-incubated with sperm (1–2 × 10⁶ sperm/mL) for 18–20 h. From MSTN-/PRNP- sperm, a total of 340 zygotes were cultured, and 88 blastocysts were formed (27.6 ± 9.9%). From MSTN-/BLG- sperm, a total of 348 zygotes were cultured and 98 blastocysts were formed (26.2 ± 7.0%) and analysed. Embryos formed from MSTN-/PRNP- sperm showed 80.6 ± 9.9% of MSTN mutation with 36.5 ± 9.1% of PRNP mutation and 23.5 ± 7.4% of mutation for both genes. From MSTN-/BLG- sperm, 37.8 ± 10.7% of embryos showed MSTN mutation and 53.1 ± 9.3% of embryos showed BLG mutation, with 21.1 ± 5.7% of embryos showing mutation for both genes. In conclusion, we proved stable germline transmission of mutations induced by the electroporation method. Those mutations did not affect the fertility of germ cells. These results show value of double gene edited bulls as valuable resources to spread multiple edited genes and therefore to boost precision breeding.

This work was supported by the Technology Innovation Program (20023353, Development of composite formulation with a sustained release (gene) for the treatment of companion animal sarcopenia) funded by the Ministry of Trade, Industry and Energy (MOTIE, Korea) and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2021R1F1A1051953).