Establishment and Application of FecB Gene Editing Method for Electrotransfer of Sheep Embryos
MA Xiu-Ling1,2,3, CHEN Ying1,2, LIN Jia-Peng1,2, WANG Li-Qin1,2, GULIMIRE•Abudureyimu1,2, WU Xiang-Li1,2, HUANG Jun-Cheng1,2, WANG Xu-Guang3,*, WU Yang-Sheng1,2,*
1 Key Laboratory of Genetics Breeding and Reproduction of Grass Feeding Livestock, Ministry of Agriculture and Rural affairs, Urumqi 830011, China; 2 Key Laboratory of Animal Biotechnology of XinJiang, Urumqi 830011, China; 3 College of Animal Sciences, XinJiang Agricultural University, Urumqi 830052, China
Abstract：FecB (fecundity booroola, FecB) gene is one of the multifetal major genes of Ovis aries, which is a single-base mutation form in the coding region of bone morphogenetic protein receptor type 1B gene. Sheep with FecB gene can be produced by gene editing technology. Gene-edited sheep are usually produced by microinjecting gene-edited material into a fertilized egg followed by transplantation to obtain offspring. Electrotransfer is a simple and easy method to introduce gene-edited materials into cells. This study aimed to establish an electrotransfer-based FecB gene editing method for sheep embryos. Gene editing materials such as Cas9 nuclease, small guide RNA (sgRNA), single stranded DNA (ssODN) and inhibitor Scr7 were used and the effects of different voltages, different time periods and different post-fertilization times on sheep embryo development and editing efficiency were compared. The results showed that the embryo had a higher survival rate and editing efficiency when the electrotransfer parameters were set at voltage of 40 V, duration of 2 ms and 4~6 h after fertilization with 5 repetitions. The overall editing efficiency reached 88%, and the percentage of embryos with point mutation was up to 60%. After transplantation of electrotransferred embryos, 3 FecB gene-edited lambs were obtained. In summary, this study established a simple, economical and efficient gene editing method for sheep fertilized eggs based on electrotransfer, which provided technical support for accelerating the promotion and application of sheep genome editing breeding and improving reproductive performance.
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