Generation of gene-edited sheep with a defined Booroola fecundity gene (FecBB) mutation in bone morphogenetic protein receptor type 1B (BMPR1B) via clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9
Shiwei Zhou A , Honghao Yu C , Xiaoe Zhao B , Bei Cai A , Qiang Ding A , Yu Huang A , Yaxin Li A , Yan Li A , Yiyuan Niu A , Anmin Lei B , Qifang Kou D , Xingxu Huang E , Björn Petersen F , Baohua Ma B , Yulin Chen A and Xiaolong Wang A G
+ Author Affiliations
- Author Affiliations
A College of Animal Science and Technology, Northwest A&F University, Yangling, 712100, China.
B College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China.
C Guilin Medical University, Guilin 541004, China.
D Ningxia Tianyuan Sheep Farm, Hongsibu, 751999, China.
E School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.
F Institute of Farm Animal Genetics, Friedrich Loeffler Institute, Neustadt 31535, Germany.
G Corresponding author. Email: xiaolongwang@nwafu.edu.cn
Reproduction, Fertility and Development 30(12) 1616-1621 https://doi.org/10.1071/RD18086
Submitted: 7 March 2018 Accepted: 3 May 2018 Published: 7 June 2018
Abstract
Since its emergence, the clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated (Cas) 9 system has been increasingly used to generate animals for economically important traits. However, most CRISPR/Cas9 applications have been focused on non-homologous end joining, which results in base deletions and insertions, leading to a functional knockout of the targeted gene. The Booroola fecundity gene (FecBB) mutation (p.Q249R) in bone morphogenetic protein receptor type 1B (BMPR1B) has been demonstrated to exert a profound effect on fecundity in many breeds of sheep. In the present study, we successfully obtained lambs with defined point mutations resulting in a p.249Q > R substitution through the coinjection of Cas9 mRNA, a single guide RNA and single-stranded DNA oligonucleotides into Tan sheep zygotes. In the newborn lambs, the observed efficiency of the single nucleotide exchange was as high as 23.8%. We believe that our findings will contribute to improved reproduction traits in sheep, as well as to the generation of defined point mutations in other large animals.
Additional keywords: amino acid exchange, genome editing, reproduction traits.
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