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
2018
Zhou, Shiwei | Yu, Honghao | Zhao, Xiaoe | Cai, Bei | Ding, Qiang | Huang, Yu | Li, Yaxin | Li, Yan | Niu, Yiyuan | Lei, Anmin | Kou, Qifang | Huang, Xingxu | Petersen, Björn | Ma, Baohua | Chen, Yulin | Wang, Xiaolong
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.
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