Shiwei Zhou

Background: After domestication, the evolution of phenotypically-varied sheep breeds has generated rich biodiversity. This wide phenotypic variation arises as a result of hidden genomic changes that range from a single nucleotide to several thousands of nucleotides. Thus, it is of interest and significance to reveal and understand the genomic changes underlying the phenotypic variation of sheep breeds in order to drive selection towards economically important traits. Review: Various traits contribute to the emergence of variation in sheep phenotypic characteristics, including coat color, horns, tail, wool, ears, udder, vertebrae, among others. The genes that determine most of these phenotypic traits have been investigated, which has generated knowledge regarding the genetic determinism of several agriculturally-relevant traits in sheep. In this review, we discuss the genomic knowledge that has emerged in the past few decades regarding the phenotypic traits in sheep, and our ultimate aim is to encourage its practical application in sheep breeding. In addition, in order to expand the current understanding of the sheep genome, we shed light on research gaps that require further investigation. Conclusions: Although significant research efforts have been conducted in the past few decades, several aspects of the sheep genome remain unexplored. For the full utilization of the current knowledge of the sheep genome, a wide practical application is still required in order to boost sheep productive performance and contribute to the generation of improved sheep breeds. The accumulated knowledge on the sheep genome will help advance and strengthen sheep breeding programs to face future challenges in the sector, such as climate change, global human population growth, and the increasing demand for products of animal origin.

Background: CRISPR/Cas9-based genome-editing systems have been used to efficiently engineer livestock species with precise genetic alterations intended for biomedical and agricultural applications. Previously, we have successfully generated gene-edited sheep and goats via one-cell-stage embryonic microinjection of a Cas9 mRNA and single-guide RNAs (sgRNAs) mixture. However, most gene-edited animals produced using this approach were heterozygotes. Additionally, non-homozygous gene-editing outcomes may not fully generate the desired phenotype in an efficient manner. Results: We report the optimization of a Cas9 mRNA-sgRNA delivery system to efficiently generate homozygous myostatin (MSTN) knockout sheep for improved growth and meat production. Firstly, an sgRNA selection software (sgRNAcas9) was used to preliminarily screen for highly efficient sgRNAs. Ten sgRNAs targeting the MSTN gene were selected and validated in vitro using sheep fibroblast cells. Four out of ten sgRNAs (two in exon 1 and two in exon 2) showed a targeting efficiency > 50%. To determine the optimal CRISPR/Cas9 microinjection concentration, four levels of Cas9 mRNA and three levels of sgRNAs in mixtures were injected into sheep embryos. Microinjection of 100 ng/μL Cas9 mRNA and 200 ng/μL sgRNAs resulted in the most improved targeting efficiency. Additionally, using both the highly efficient sgRNAs and the optimal microinjection concentration, MSTN-knockout sheep were generated with approximately 50% targeting efficiency, reaching a homozygous knockout efficiency of 25%. Growth rate and meat quality of MSTN-edited lambs were also investigated. MSTN-knockout lambs exhibited increased body weight and average daily gain. Moreover, pH, drip loss, intramuscular fat, crude protein, and shear force of gluteal muscles of MSTN-knockout lambs did not show changes compared to the wild-type lambs. Conclusions: This study highlights the importance of in vitro evaluation for the optimization of sgRNAs and microinjection dosage of gene editing reagents. This approach enabled efficient engineering of homozygous knockout sheep. Additionally, this study confirms that MSTN-knockout lambs does not negatively impact meat quality, thus supporting the adoption of gene editing as tool to improve productivity of farm animals.