Production Performance And Molecular Biological Evaluation Of MSTN Gene Knockout Cashmere Goats

The recently emerging genetic engineering tools(such as CRISPR-based genome editing systems)has enabled a precise targeting of animal and plant genomes.In animal breeding,genome editing technologies can greatly shorten the breeding years and improve the production performance of livestock and poultry.Myostatin(MSTN),known as the "superstar gene",negatively regulates animal muscle development and growth,thereby it can be targeted to improve meat yield and quality.In our laboratory,a number of MSTN gene-edited cashmere goats were obtained using CRISPR/Cas9 technology,however,the systematic molecular biological evaluation of the gene-edited animals is lacking.In this study,MSTN gene knockout Shaanbei white cashmere goats were used.We applied different methods to detect the production traits of gene-edited goats and used proteomic and metabolomic analyses of muscle tissues to evaluate meat and growth traits at molecular biological level.The measurements of production traits include blood biochemical indicators,body size,slaughter performance,meat quality,and tissue morphology analyses.Molecular biological evaluation includes the analysis of gene editing efficiency using targeted deep sequencing,as well as proteomic and metabolomic analyses of muscles using isobaric tags for relative and absolute quantitation(i TRAQ)and liquid chromatography tandem mass spectrometry(LC-MS)were used.In this study,four MSTN gene knockout goats(#02,#30,#34,and #40)were used as the experimental group,and three wild-type goats(#08,#12,and #14)were used as the control group.The main results obtained in this study are as follows:1.The determination of production traits and morphological observation of tissue sections.There was no significant difference in the morphological structure between the experimental and control groups,whereas,the longitudinal section area of muscle fiber,body weight,slaughter rate were significantly increased(P<0.05).These indicate the improved body size and increased meat production of the experimental group.The muscle tenderness was significantly reduced(P<0.05),which indicates a potential change of the palatability.2.Evaluation of MSTN gene knockout goats.The targeted deep sequencing of muscle tissues in the experimental group showed that all the four goats were gene-edited and the editing outcomes included various degrees of deletions and insertions.The highest editing efficiency was 97.81%.3.Proteomic analysis of muscle tissues.Through proteomic analysis of gluteal muscles between the two groups,we identified 25 proteins with significantly different expression levels.Among them,7 proteins were upregulated and 18 were downregulated.Most of these differentially expressed proteins are located in the cytoplasmic matrix and the domain of protein has a binding function with ATP.Gene Ontology(GO)analysis showed that the cellular composition of the differentially expressed proteins was enriched in the vacuole membrane and the cytoplasmic membrane.The molecular function was mainly to combine with small molecules on synapses and biological processes were involved in hormone secretion and signal transmission.4.Metabolomic analysis of muscle tissues.Through metabolomic analysis of the gluteal muscles between the two groups,we found that 13 metabolites were significantly differentially expressed.Of these,3 metabolites were increased and 10 metabolites were decreased.Kyoto encyclopedia of genes and genomes(KEGG)pathway analysis showed that these metabolites mainly participate in the body energy synthesis and metabolism,including amino acid synthesis,lipid metabolism,and pentose phosphate pathway.In summary,the MSTN gene knockout in goats affected the physiological activities related to energy metabolism and improved meat production and growth traits.Additionally,the rest of the body functions were not affected.The energy and proteins provided by the body is inseparable to the muscle growth.The muscle growth of the MSTN gene knockout goats was accelerated and the demand for energy and protein was higher,which affected physiological activities that are related to energy and protein metabolism.In this study,the production traits,muscle proteomics,and metabolomics of MSTN gene knockout goats were analyzed,and the results showed that the MSTN gene knocking out promotes the growth of goat muscles and affects the body protein and energy metabolism pathways.This study promotes the application of genome editing technology and provides a reference for the establishment of the safety evaluation system of gene-edited animals.