161 Germline transmission of MSTN knockout cattle via CRISPR-Cas9
G.-M. Kim A C , K.-H. Uhm A , D.-H. Kwon A , M.-J. Kim A , D.-J. Jung B , D.-H. Kim B , J.-K. Yi B , J.-J. Ha B , S.-Y. Yum C , W.-J. Son C , J.-H. Lee C , K.-Y. Song C , W.-W. Lee C and G. Jang A CA College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
B Gyeongsangbukdo Livestock Research Institute, YeongJu, Republic of Korea
C LARTBio Inco., Seoul, Republic of Korea
Reproduction, Fertility and Development 35(2) 208-208 https://doi.org/10.1071/RDv35n2Ab161
Published: 5 December 2022
© 2023 The Author(s) (or their employer(s)). Published by CSIRO Publishing on behalf of the IETS
Although the production of several founder animals (F0) for gene editing in livestock has been reported in cattle, very few studies have assessed germline transmission to the next generation due to long-sexual maturity and -gestation period. In previous our study, we produced two MSTN mutant male and female using CRISPR-Cas9, with 10.5% and 99.9% MSTN mutation, respectively. The present study aimed to assess the germline transmission of MSTN mutations in MSTN mutant F0. For this purpose, oocytes and semen were collected after sexual maturation of MSTN cattle and produced embryo by in vitro production system was analysed. In addition, for further gene editing (PRNP), Cas9 protein, and sgRNA were introduced into zygotes using electroporation. As a result, using ovum pickup, 66 oocytes were recovered from MSTN female, and their blastocyst formation rate was 13.0 ± 9.8%. The germline transmission of MSTN mutations (−12 bp deletion) in their blastocyst was observed. Seven blastocysts produced by in vitro production with MSTN F0 male and female were transferred to seven surrogates, and one calf was successfully born. F1 calf showed MSTN heterologous mutation through sequencing, and no health issues. As a further experiment to additional gene editing using electroporation, 336 oocytes were fertilised with MSTN male mutant F0 germ cells, and 99 blastocysts (26.0 ± 0.5%) were produced. Electroporated embryos showed that PRNP mutation was 86.2 ± 3.4%, MSTN mutation was 7.7 ± 4.0, and double mutation (PRNP and MSTN) was 6.0 ± 3.6. These data demonstrate that the cattle through gene editing could be grown up to date without health issues, transmitted MSTN mutation to germ cells for the next generation, and additional gene editing of embryos derived from F0 semen could be possible for further mutagenesis using electroporation.
This study was financially supported by the National Research Foundation of Korea (2017R1A2B3004972), the Research Institute of Veterinary Science, the BK21 Four for Future Veterinary Medicine Leading Education and Research Center, and a Seoul National University (SNU) grant (#550-2020005). We thank the members of the Goo Jang laboratory and the National Agricultural Cooperative Federation Bucheon Livestock Market for their valuable comments and the technical support.
