RESEARCH ARTICLE Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and

While CRISPR/Cas9 technology has proven to be a valuable system to generate gene-targeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded byMSTN gene involved in the inhibition of muscle differenti-ation and growth. We determined the efficiency of the CRISPR/Cas9 system to editMSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle develop-ment and body growth. We generated CRISPR/Cas9 mRNAs specific for ovineMSTN and microinjected them into the cytoplasm of ovine zygotes. When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both micro-injected groups (P<0.05) and neither was affected by CRISPR/Cas9 content in the injected medium. Embryo development to blastocyst was not affected by microinjection and was similar among the experimental groups. From 20 embryos analyzed by Sanger sequencing, ten were mutant (heterozygous or mosaic; 50 % efficiency). To obtain liveMSTN KO lambs, 53 blastocysts produced after zygote CRISPR/Cas9 microinjection were transferred t