Generation of double knockout cattle via CRISPR-Cas9 ribonucleoprotein (RNP) electroporation

Journal of Animal Science and Biotechnology

Research

Dae-Jin Jung¹  Dae-Hyun Kim¹  Jae-Jung Ha¹  Jun-Koo Yi¹  Ji-Hyun Lee²  Won-Wu Lee²  Seong-Beom Lee²  Soo-Young Yum²  Woo-Jae Son²  Dong-Hyeok Kwon³  Kyeong-Hyeon Eom⁴  Gyeong-Min Gim⁴  Goo Jang⁵ 

[1] Gyeongsangbukdo Livestock Research Institute, Yeongju, Republic of Korea;LARTBio Co., Ltd., Seoul, Republic of Korea;Laboratory 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;Laboratory 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;LARTBio Co., Ltd., Seoul, Republic of Korea;Laboratory 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;LARTBio Co., Ltd., Seoul, Republic of Korea;Comparative Medicine Disease Research Center, Seoul National University, Seoul, Republic of Korea;

关键词: Beta-lactoglobulin, Cattle;    CRISPR-Cas9;    Electroporation;    Knockout;    MSTN;    PRNP;   

DOI  :  10.1186/s40104-023-00902-8

 received in 2023-02-13, accepted in 2023-06-04,  发布年份 2023

来源: Springer

【 摘 要 】

BackgroundGenome 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.ResultsFirst, 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.ConclusionsThese 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.

【 授权许可】

CC BY   
© The Author(s) 2023

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