CRISPR/Cas9‐mediated MSTN disruption and heritable mutagenesis in goats causes increased body mass

Abstract

Genetic engineering in livestock has been greatly enhanced through the use of artificial programmed nucleases such as the recently emerged clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system. We recently reported our successful application of the CRISPR/Cas9 system to engineer the goat genome through micro-injection of Cas9 mRNA and sgRNAs targeting MSTN and FGF5 in goat embryos. The phenotypes induced by edited loss-of-function mutations of MSTN remain to be evaluated extensively. We demonstrate the utility of this approach by disrupting MSTN, resulting in enhanced body weight and larger muscle fiber size in Cas9-mediated gene-modified goats. The effects of genome modifications were further characterized by H&E staining, quantitative PCR, Western blotting and immunofluorescence staining. Morphological and genetic analyses indicated the occurrence of phenotypic and genotypic modifications. We further provide sufficient evidence, including breeding data, to demonstrate the transmission of the knockout alleles through the germline. By phenotypic and genotypic characterization, we demonstrated the merit of using the CRISPR/Cas9 approach for establishing genetically modified livestock with an enhanced production trait.

Muscle Hyperplasia in Japanese Quail by Single Amino Acid Deletion in MSTN Propeptide

“…In most terrestrial vertebrates, muscle fiber number is known to be fixed before birth [41,42] and expression of MSTN is observed during embryonic stage when muscle fiber is developed [2,43]. Thus, MSTN knockout can affect muscle development from the embryonic stage and increased fetal and birth weight of MSTN mutant animals was observed in sheep, cattle, and goats [9,34,44]. In mammals, nutrients are continuously supplied through maternal circulation during embryonic development, which can further support enhanced muscle growth by MSTN mutation in fetus.…”

CRISPR in livestock: From editing to printing

“…The knockout lambs were 25% heavier [7] and maintained the same wool quality traits than Merino lambs (unpublished data). Although this proof of concept did not include the study of the offspring, in following studies germline transmission of MSTN mutation have been reported by its presence in the gonads of founders (in sheep [46] and goats [47]) and offspring [47]. Amazingly, these early studies suggested that what farmers have pursued for centuries (i.e., animals producing both high-quality wool and more meat), might be achieved by CRISPR in few months.…”

Themyostatingene: an overview of mechanisms of action and its relevance to livestock animals

“…Wang et al . () reported the successful application of the CRISPR/Cas9 system to engineer the goat genome through micro‐injection of Cas9 mRNA and sgRNAs targeting MSTN in goat embryos. They demonstrated the utility of this approach by disrupting MSTN , resulting in enhanced body weight and larger muscle fiber size in Cas9‐mediated gene modified goats.…”