Abstract
Myostatin (Mstn) is a conserved negative regulator of skeletal muscle mass in mammals. However, whether precise disruption of Mstn in livestock can be achieved and safely used to improve meat productivity has not been proven. We applied CRISPR/Cas9 system to generate Mstn knock-out (KO) rabbits and goats and then analyzed the changes in their phenotypes to answer this question. We efficiently generated 24 Mstn KO rabbits out of 32 newborn infants after embryo injection with two sgRNAs targeting rabbit Mstn, and found that the Mstn KO rabbits exhibited increased birthweight and a significantly increase in the weight ratios of the quadriceps and biceps muscles to the whole body. Mstn KO also caused high probability of enlarged tongue phenomenon and severe health problems such as stillbirth and early stage death. Using the same method, one out of four goats was generated with edition at Mstn locus. The early stage growth rate of this goat outperformed the control goats. In conclusion, we efficiently generated Mstn KO rabbits and goats using CRISPR/Cas9 technology. However, Mstn KO causes severe health problems and may also have the same effects on other species. This safety issue must be studied further before applied to animal reproduction processes.
Highlights
Myostatin (Mstn), known as growth differentiation factor 8 (GDF-8), belongs to the TGF-beta superfamily
In this study, we utilized CRISPR/Cas[9] system together with cytoplasm injection method to knock out Mstn in rabbit and goat, and we studied the changes in the phenotypes and early stage development of Mstn KO rabbits and goats
We first studied whether CRISPR/Cas[9] can be used efficiently at the Mstn locus in rabbit embryos
Summary
We first studied whether CRISPR/Cas[9] can be used efficiently at the Mstn locus in rabbit embryos. Twenty-six one-cell stage rabbit embryos were injected with a mixture of 200 ng/μl Cas[9] mRNA and 20 ng/μl sgR1 or sgR2 (Table S3). 14 out of 26 injected embryos developed to the morula or blastocyst stages (Table S3) These embryos were collected and examined for the presence of site-specific genome modification using T7E1 cleavage assay and Sanger sequencing. During the early infant stage, 8 more rabbits died due to weakness or were euthanized because of their poor health condition. Their tongues and other tissues were collected for further analysis. T7E1 cleavage assay was used to for the AF an infant #1-#5 (Fig. 2b,c), and direct Sanger sequencing of the PCR amplicons of the sgR1 and sgR2 sites were used for the remaining 29 infants to detect genome editing
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