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

作　　者：Gyeong-Min Gim Kyeong-Hyeon Eom Dong-Hyeok Kwon Dae-Jin Jung Dae-Hyun Kim Jun-Koo Yi Jae-Jung Ha Ji-Hyun Lee Seong-Beom Lee Woo-Jae Son Soo-Young Yum Won-Wu Lee Goo Jang 

机构地区：[1]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 [2]LARTBio Co.,Ltd.,Seoul,Republic of Korea [3]Gyeongsangbukdo Livestock Research Institute,Yeongju,Republic of Korea [4]Comparative Medicine Disease Research Center,Seoul National University,Seoul,Republic of Korea

出　　处：《Journal of Animal Science and Biotechnology》2024年第1期456-462,共7页畜牧与生物技术杂志（英文版）

基　　金：financially supported by the National Research Foundation of Korea(NRF-2021R1A5A1033157 for SRC program:382 Comparative medicine Disease Research Center,NRF-2021R1F1A105195313);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(#550e2020005)。

摘　　要：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.

关 键 词：BETA-LACTOGLOBULIN CATTLE CRISPR-Cas9 ELECTROPORATION KNOCKOUT MSTN PRNP 

分 类 号：S823[农业科学—畜牧学] Q78[农业科学—畜牧兽医]