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.