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Myostatin acts as a negative regulator of muscle growth; therefore, its role is important with regard to animal growth and meat production. This study was undertaken with the objective to detect polymorphisms in the first intron and c.*1232 position of the MSTN gene and to analyze effects of the detected alleles/genotypes on growth and carcass traits in Colored Polish Merino sheep. In total, 23 traits were analyzed, i.e., seven describing lamb growth and 16 carcass traits. Single nucleotide polymorphisms (SNPs) in the first intron and the c.*1232 position were identified using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and PCR-restriction fragment length polymorphism (PCR-RFLP) methods, respectively. The MIXED procedure of the SAS software package was used to analyze allelic and genotypic effects of the MSTN gene on growth and carcass traits. Polymorphisms were only detected in the first intron of the MSTN gene. All investigated sheep were monomorphic G in the c.*1232 position. The MSTN genotype was found to have significant effect on body weight at 2nd day of life (BW2) and loin and fore shank weights. Significant allelic effects were detected with respect to BW2, scrag, leg, fore, and hind shank weights. These results suggest that polymorphisms in the first intron of the MSTN gene are relevant with respect to several carcass traits and BW2 in Colored Polish Merino sheep.

Polymorphisms of myostatin ( MSTN ) gene were investigated as a candidate marker for goat growth in 687 individuals by gene sequencing and polymerase chain reaction restriction fragment length polymorphism methods. Three potential genotypes (AA, AB and BB) of 5 bp indel (1,256 TTTTA/−) in 5′UTR were detected in four breeds. The polymorphism (CC, CD and DD) of substitution (1,388 T/A) in exon 1 was only segregating in Boer. Genotype AB resulted in significant increases in body weights at birth (BW0), 90 (BW90) and 300 (BW300) days of age, and birth body length compared to BB ( P  < 0.05). Whilst genotype CD contributed to heavier BW0 and BW90, and larger birth body height (BH) compared to CC ( P  < 0.05). Individuals either with AB or CD genotype also had greater values in other body sizes, although no significant differences appeared ( P  > 0.05). When in combination, the combined genotype ABCD displayed positive impacts on better growth traits in BW0, BW90, BW300, BH and BCG ( P  < 0.05). These results indicate that these markers in myostatin ( MSTN ) are associated with Boer growth and may be useful for marker assisted selection.

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.

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.

Chinese Yellow Cattle, an ancient and domesticated breed for draft service, provide unique animal genetic resources with excellent genetic features, including crude feed tolerance, good stress resistance, strong adaptability, and tender meat quality; however, their production performance and meat yield are significantly inferior. Herein, the myostatin gene (MSTN), a negative regulator of skeletal muscle development, was knocked out by CRISPR/Cas9 technology. Eight MSTN gene-edited bull calves (MT) were born, and six of them are well-developed. Compared with the control cattle (WT), the growth trait indexes of MT cattle were generally increased, and the hindquarters especially were significantly improved. The biochemical indexes and the semen characteristics demonstrated that MT bulls were healthy and fertile. Consistent with our conjecture, the wobble and beating of MT bull spermatozoa were significantly higher than that of WT. Nine sperm motility-related proteins and nineteen mitochondrial-related proteins were identified by up-regulation in MT bull spermatozoa using FLQ proteomic technique and act to govern sperm flagellum assembly, organization, and beating and provide sufficient energy for sperm motility. The current study confirmed that the MSTN gene-edited Chinese Yellow cattle have improved growth traits and normal fertility, which can be used for beef cattle production and breeding.

Myostatin (MSTN) is a dominant inhibitor of skeletal muscle development and growth. Mutations in MSTN gene can lead to muscle hypertrophy or double-muscled (DM) phenotype in cattle, sheep, dog and human. However, there has not been reported significant muscle phenotypes in pigs in association with MSTN mutations. Pigs are an important source of meat production, as well as serve as a preferred animal model for the studies of human disease. To study the impacts of MSTN mutations on skeletal muscle growth in pigs, we generated MSTN -mutant Meishan pigs with no marker gene via zinc finger nucleases (ZFN) technology. The MSTN- mutant pigs developed and grew normally, had increased muscle mass with decreased fat accumulation compared with wild type pigs and homozygote MSTN mutant ( MSTN −/− ) pigs had apparent DM phenotype and individual muscle mass increased by 100% over their wild-type controls ( MSTN +/+ ) at eight months of age as a result of myofiber hyperplasia. Interestingly, 20% MSTN- mutant pigs had one extra thoracic vertebra. The MSTN- mutant pigs will not only offer a way of fast genetic improvement of lean meat for local fat-type indigenous pig breeds, but also serve as an important large animal model for biomedical studies of musculoskeletal formation, development and diseases.

Gene therapy is a promising method for treating inherited diseases by directly delivering the correct genetic material into patient cells. However, the limited packaging capacity of vectors poses a challenge. Minimizing promoter size is a viable strategy among various approaches to address this issue. This study aims to optimize the bovine myostatin ( MSTN ) promoter, enhancing its utility in gene therapy applications. We identified the primary driver of activity as the proximal regulatory region. Isolated from the native promoter and termed M243, this 243-bp sequence was assessed for its potential as a ubiquitous promoter. In a variety of cell types, including bovine embryos and embryonic stem cells, the M243 promoter showed consistent expression, highlighting its suitability for applications requiring compact promoters. The isolation of a highly conserved, compact 243-bp sequence serving as a promoter suggests a solution for overcoming the size constraints of AAV vectors, suggesting potential contributions to the field of gene therapy.

The knockout (KO) of the myostatin (MSTN) gene can increase muscle production in Mongolian cattle; however, the safety of MSTN-KO beef has not been evaluated. In this study, we fed mice varying concentrations of MSTN-KO beef and monitored physiological and tissue changes. Compared with the control group fed with wild-type beef, mice fed with MSTN-KO beef did not show significant changes, including weight gain, food intake, and organ weight. Furthermore, most blood parameters of the experimental groups remained stable. Serum metabolomics analysis confirmed that MSTN-KO beef had a limited impact on the mice’s overall metabolism, with only 24 differential metabolites identified. Our findings from this 90-day trial show no toxic effects of MSTN-KO Mongolian beef on mice. This directly addresses the long-standing lack of toxicity data for such gene-edited beef. Notably, this is the first study to fill the research gap, and the evidence generated in this work actively supports the safety assessment of MSTN-modified animal-derived foods.

Predictable, clean genetic modification (GM) in livestock is important for reliable phenotyping and biosafety. Here we reported the generation of isozygous, functional myostatin (MSTN) knockout cloned pigs free of selectable marker gene (SMG) by CRISPR/Cas9 and Cre/LoxP. CRISPR/Cas9-mediated homologous recombination (HR) was exploited to knock out (KO) one allele of MSTN in pig primary cells. Cre recombinase was then used to excise the SMG with an efficiency of 82.7%. The SMG-free non-EGFP cells were isolated by flow cytometery and immediately used as donor nuclei for nuclear transfer. A total of 685 reconstructed embryos were transferred into three surrogates with one delivering two male live piglets. Molecular testing verified the mono-allelic MSTN KO and SMG deletion in these cloned pigs. Western blots showed approximately 50% decrease in MSTN and concurrent increased expression of myogenic genes in muscle. Histological examination revealed the enhanced myofiber quantity but myofiber size remained unaltered. Ultrasonic detection showed the increased longissimus muscle size and decreased backfat thickness. Precision editing of pig MSTN gene has generated isozygous, SMG-free MSTN KO cloned founders, which guaranteed a reliable route for elite livestock production and a strategy to minimize potential biological risks.

As gene-editing technologies continue to evolve, gene-edited products are making significant strides. These products have already been commercialized in the United States and Japan, prompting global attention to their safety and regulatory oversight. However, the detection of gene editing still relies on qPCR, and there is a lack of quantitative detection methods to quantify gene-editing components in products. To ensure consumer safety and transparency, we developed a novel droplet digital PCR (ddPCR)-based detection method for gene-edited products. Primers and probes were designed targeting the editing sites of MSTN-edited cattle, and the method was evaluated for specificity, sensitivity, real sample testing, and detection thresholds. Our results demonstrate that this ddPCR method is highly specific, with a detection limit of 5 copies/µL, and it successfully detected MSTN edits in all 11 tested samples. Tests using both actual gene-edited cattle samples and plasmid DNA at concentrations of 5%, 1%, and 0.01% yielded consistent results, indicating the method’s suitability for real-world applications. This ddPCR assay provides a sensitive and specific tool for detecting MSTN gene-edited cattle and determining the presence of gene-edited products, offering crucial support for regulatory monitoring of gene-edited animal-derived foods.