Abstract
Many transgenic animals have been produced using CRISPR–Cas9 technology to edit specific genes. However, there are few guidelines for the application of this technique in cattle. The goal of this study was to produce trait-improved cattle using the genome editing technology CRISPR–Cas9. Myostatin (MSTN ) was selected as a target locus and synthetic mRNA of sgRNA and Cas9 was microinjected into bovine in vitro fertilized embryos. As a result, 17 healthy calves were born and 3 of these showed MSTN mutation rates of 10.5%, 45.4%, and 99.9%, respectively. Importantly, the offspring with the 99.9% MSTNmutation rate had biallelic mutation (-12bp) and a doubling muscle growth phenotype. In conclusion, we showed that the genome editing technology CRISPR–Cas9 can produce genetically modified calves with improved traits.
Many animal product (milk and meat) studies focus on the improvement of performance traits in cattle because cattle contribute 45% of the global animal protein supply for human consumption [1,2]. Significant effort has been made to improve the trait of cattle using advanced reproductive technologies (ART) [3,4] based on genotyping and phenotyping analysis. One application of genotyping and phenotyping breeding is to select and propagate the breeds with high amount of muscle. Belgian Blue and Piedmontese are the most representative double muscles cattle breeds. [5]. Genetic analysis identified the mutation of MSTN (growth and differentiation factor 8) as the causative factor for enhanced muscle development. Mutations in the gene have also been observed in the dog [6], sheep [7], pig [8] and human [9]. However, the incidence of these natural mutations is very low and selecting and breeding these individuals to establish an independent breed is time-consuming and costly.
The development of genome editing tools such as zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR–Cas9) has provided new and powerful gene editing tools for functional mutation studies in various biotechnological industries. Applying these genome editing tools to livestock will contribute to: improvements in cattle traits, and better understanding of how to prevent and treat cattle disease [10]. Proof of concept gene edited cattle—using bovine somatic cell nuclear transfer (SCNT)—have been produced with enhanced traits for disease resistance, allergen removal, production, and welfare [11-15]. Because SCNT is used to produce cloned offspring with precise gene edited cells, it is employed in cattle to generate a valuable model [16,17]. However, abnormal reprogramming results in low efficiency and survival rate, limiting the application of SCNT. An alternative approach to increase the efficiency of gene editing is through Belgian Blue the microinjection of in vitro fertilized embryos [18]. One study used TALENs for editing MSTN gene microinjection of in vitro fertilized cattle embryos [19]. However, there are no reports of a live-born genome edited calf using CRISPR–Cas9 on the MSTN locus. Accordingly, the aim of present study is 1) to develop a method to produce gene edited bovine pre-implantation embryos using through microinjection with Cas9 mRNA and 2) to produce the MSTN edited calves.