Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes

Abstract

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 by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN 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 microinjected 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 live MSTN KO lambs, 53 blastocysts produced after zygote CRISPR/Cas9 microinjection were transferred to 29 recipient females resulting in 65.5% (19/29) of pregnant ewes and 41.5% (22/53) of newborns. From 22 born lambs analyzed by T7EI and Sanger sequencing, ten showed indel mutations at MSTN gene. Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons. Western blot analysis of homozygous KO founders confirmed the absence of myostatin, showing heavier body weight than wild type counterparts. In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep. This technology is quick and easy to perform and less expensive than previous techniques, and can be applied to obtain genetically modified animal models of interest for biomedicine and livestock.

Genome Editing with Engineered Nucleases in Economically Important Animals and Plants: State of the Art in the Research Pipeline

“…The knock-out of the mstn gene thus results in a substantial increase of skeletal muscle mass and in increased meat production. Using SDNs, the gene was artificially mutated in cattle Proudfoot et al, 2015) and pigs (Huang et al, 2014b;Qian et al, 2015;Wang et al, 2015b), but also in smaller livestock species like sheep Zhang et al, 2014a;Crispo et al, 2015;Proudfoot et al, 2015) and goat (Ni et al, 2014;Wang et al, 2015c). In most cases, the studies have chosen to target the first exon of the gene and obtained small deletions and insertions in the gene.…”

“…In most cases, the studies have chosen to target the first exon of the gene and obtained small deletions and insertions in the gene. In the studies of Proudfoot et al (2015), Han et al (2014), Luo et al (2014), Crispo et al (2015), Wang et al (2015c), and Wang et al (2015a) several offspring were born that had the desired mstn knock-out. The animals were visually different from the wild type individuals with an obvious difference in muscle mass and a difference was also observed during histological comparison of the muscles Qian et al, 2015).…”

Transcriptome Analysis of Non‐Coding RNAs in Livestock Species: Elucidating the Ambiguity

“…In the catle industry, gene-edited calves have been produced with speciied genetics by ovum pickup, in vitro fertilization and zygote microinjection (OPU-IVF-ZM). The CRISPR/Cas9 system has also been used eiciently to generate gene knock out sheep [224].…”

Redesigning Nature:To Be or not to Be?

“…The process has already been used on cows and sheep with 50% success rate 15 . The CRISPR/Cas9 system has been used to modify three genetic loci in Bama pigs: parkin, DJ-1 and PINK1 in order to study Parkinson's disease 16 .…”