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J. Anim. Sci. Biotechnol., 2023 Aug 6;14(1):103 | https://doi.org/10.1186/s40104-023-00902-8 Generation of double knockout cattle via CRISPR-Cas9 ribonucleoprotein (RNP) electroporation

Gyeong‑Min Gim^1,2†, Kyeong‑Hyeon Eom^1,2†, Dong‑Hyeok Kwon¹, Dae‑Jin Jung³, Dae‑Hyun Kim3, Jun‑Koo Yi³, Jae‑Jung Ha³, Ji‑Hyun Lee², Seong‑Beom Lee², Woo‑Jae Son², Soo‑Young Yum², Won‑Wu Lee² and Goo Jang^1,2,4*

1Laboratory of Theriogenology and Biotechnology, Department of Veterinary Clinical Science, College of Veterinary Medicine and the Research Institute of Veterinary Science, Seoul National University, Seoul, Republic of Korea
2LARTBio Co., Ltd., Seoul, Republic of Korea
3Gyeongsangbukdo Livestock Research Institute, Yeongju, Republic of Korea
4Comparative Medicine Disease Research Center, Seoul National University, Seoul, Republic of Korea

^†Gyeong-Min Gim and Kyeong-Hyeon Eom contributed equally to this work.
^*Correspondence: Goo Jang

Abstract

Background: Genome editing has been considered as powerful tool in agricultural fields. However, genome editing progress in cattle has not been fast as in other mammal species, for some disadvantages including long gestational periods, single pregnancy, and high raising cost. Furthermore, technically demanding methods such as microinjection and somatic cell nuclear transfer (SCNT) are needed for gene editing in cattle. In this point of view, electroporation in embryos has been risen as an alternative.

Results: First, editing efficiency of our electroporation methods were tested for embryos. Presence of mutation on embryo was confirmed by T7E1 assay. With first combination, mutation rates for MSTN and PRNP were 57.6% ± 13.7% and 54.6% ± 13.5%, respectively. In case of MSTN/BLG, mutation rates were 83.9% ± 23.6% for MSTN, 84.5% ± 18.0% for BLG. Afterwards, the double-KO embryos were transferred to surrogates and mutation rate was identified in resultant calves by targeted deep sequencing. Thirteen recipients were transferred for MSTN/PRNP, 4 calves were delivered, and one calf underwent an induction for double KO. Ten surrogates were given double-KO embryos for MSTN/BLG, and four of the six calves that were born had mutations in both genes.

Conclusions: These data demonstrated that production of genome edited cattle via electroporation of RNP could be effectively applied. Finally, MSTN and PRNP from beef cattle and MSTN and BLG from dairy cattle have been born and they will be valuable resources for future precision breeding.

논문정보

형식Research article
게재일2023년 08월 (BRIC 등록일 2023-08-17)
연구진국내 연구진
분야 의약학 > 응용의학

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