94 Efficient generation of myostatin mutation in Korean beef cattle

Abstract

In recent years, CRISPR/Cas9 has become known as a powerful tool for generating genetically modified cells or animal models with its site-specific gene editing capability. Because myostatin (MSTN) is associated with muscle differentiation and growth, ablation of MSTN would be ideal for studying phenotypes associated with muscle growth in beef cattle. The aim of this study is to produce Korean beef cattle with MSTN mutation through microinjection of CRISPR/Cas9. For this study, ovaries of Korean beef cattle were obtained from slaughterhouses; 122 immature oocytes were aspirated from 8 Korean beef cattle. The oocytes were matured in tissue culture medium 199-based medium for 22 h and inseminated with frozen-thawed semen to produce zygotes. All 122 zygotes were directly injected with 200 ng of Cas9 mRNA and 100 ng of sgRNA for MSTN after IVF and were cultured for 7 days in two-step chemically defined medium. Each embryo from the individual cattle was cultured separately to distinguish its lineage. After 7 d, 29 (23.8 ± 8.9) blastocysts were recovered, and all blastocysts were assessed with T7 endonuclease (T7E1) mutation assay to check for mutations at the MSTN locus. Based on T7E1, 26 out of 29 blastocysts (89.7 ± 10.5%) had heterozygous mutation in the MSTN gene. After analysis, additional blastocysts were produced and transferred into 14 surrogates. All surrogates were diagnosed for pregnancy via rectal palpation and ultrasonography at Day 55, at which time normal fetuses and embryonic sacs were confirmed in 10 surrogates. In conclusion, these data demonstrate that CRISPR/Cas9 could be employed as an efficient method to genetically modify the MSTN gene in Korean beef cattle.