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

被引:14
作者
Gim, Gyeong-Min [1 ,2 ]
Eom, Kyeong-Hyeon [1 ,2 ]
Kwon, Dong-Hyeok [1 ]
Jung, Dae-Jin [3 ]
Kim, Dae-Hyun [3 ]
Yi, Jun-Koo [3 ]
Ha, Jae-Jung [3 ]
Lee, Ji-Hyun [2 ]
Lee, Seong-Beom [2 ]
Son, Woo-Jae [2 ]
Yum, Soo-Young [2 ]
Lee, Won-Wu [2 ]
Jang, Goo [1 ,2 ,4 ]
机构
[1] Seoul Natl Univ, Res Inst Vet Sci, Lab Theriogenol & Biotechnol, Dept Vet Clin Sci,Coll Vet Med, Seoul, South Korea
[2] LARTBio Co Ltd, Seoul, South Korea
[3] Gyeongsangbukdo Livestock Res Inst, Yeongju, South Korea
[4] Seoul Natl Univ, Comparat Med Dis Res Ctr, Seoul, South Korea
基金
新加坡国家研究基金会;
关键词
Beta-lactoglobulin; Cattle; CRISPR-Cas9; Electroporation; Knockout; MSTN; PRNP;
D O I
10.1186/s40104-023-00902-8
中图分类号
S8 [畜牧、 动物医学、狩猎、蚕、蜂];
学科分类号
0905 ;
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% & PLUSMN; 13.7% and 54.6% & PLUSMN; 13.5%, respectively. In case of MSTN/BLG, mutation rates were 83.9% & PLUSMN; 23.6% for MSTN, 84.5% & PLUSMN; 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.
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页数:7
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