This study successfully established a YE1-AncBE4max-based platform for efficient, precise dual-gene editing in goats and showed that the edited goats exhibited significantly increased body weight and a "double-muscling" phenotype by 90 days of age compared to wild-type controls.
Myostatin (mstn) is a negative regulator of skeletal muscle growth and is considered as an important target for enhancing aquaculture production. The present study aimed to design and validate single-guide RNAs (sgRNAs) and CRISPR/Cas9 constructs for exon 1 of the mstnb gene in Labeo rohita, and to evaluate their transfection efficiency in the L. rohita dorsal muscle (LRDM) cell line at the 10th, 20th, and 30th passages. sgRNAs were designed and cloned into the pSpCas9(BB)-2A-GFP (PX458) vector using BbsI restriction digestion and ligation. Successful insertion and correct orientation of the sgRNAs were confirmed through Sanger sequencing. LRDM cells were revived and maintained in L-15 medium supplemented with 10% fetal bovine serum. Transfection was performed at the 10th, 20th, and 30th passages. Distinct GFP-positive cells were observed at all passages for both sgRNA constructs, indicating the ability of the developed cell line to successfully express the constructs across different passages. The study successfully established CRISPR/Cas9 plasmid constructs for the mstnb gene in L. rohita and demonstrated their transfection in LRDM cell line across multiple passages. These findings provide a basis for future studies on genome editing approaches using CRISPR/Cas9 constructs in fish muscle cell lines and highlight the potential application of CRISPR/Cas9 technology for genetic engineering applications in fish muscle cells.
Introduction The rapid development of genome editing technologies has enabled precise manipulation of livestock genomes for the improvement of production traits such as meat yield and milk quality. Myostatin (MSTN) and β-lactoglobulin (BLG) are key gene targets for enhancing muscle growth and reducing lactose intolerance, respectively. Methods In this study, we employed an optimized CRISPR/EOCas12i system to simultaneously target MSTN and BLG in bovine fetal fibroblasts (BFFs) using a single plasmid. Results T7E1 and Sanger sequencing confirmed efficient editing at multiple target sites, with EOCas12i producing deletions ranging from tens to over 100 bp. Furthermore, no off-target (OT) effects were detected at predicted loci, supporting the high specificity of this system in large animals. Gene-edited single-cell clones (SCCs) were expanded in conditioned medium, and selected double-knockout (DKO) clones served as nuclear donors for somatic cell nuclear transfer (SCNT) to produce MSTN/BLG double gene-edited cattle embryos. Discussion Collectively, this study demonstrates the feasibility of generating MSTN/BLG double gene-edited cattle embryos using a single CRISPR/EOCas12i plasmid and SCNT, providing a robust platform for multiplex genome editing aimed at improving meat production and milk traits, with potential applications in both agricultural and biomedical research.
Furui Wang, Lei Chen, Yuting Ning et al.· Frontiers in Genome Editing· 0 citations
The effects of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9‐mediated disruption of the myostatin gene (mstnb1) on the growth performance of rainbow trout (Oncorhynchus mykiss) were evaluated. Two specific guide RNAs (gRNAs) targeting the first exon of the target gene were co‐injected into fertilized eggs. While the survival rate of the injected embryos at the swim‐up stage was 23.5%, Sanger sequencing of F0 individuals confirmed the successful generation of mutations. During a 10‐month growing period, quantifiable phenotypic data revealed a significant enhancement in macroscopic somatic growth. The mstnb1‐F0 mosaic fish exhibited an approximate 56% increase in body weight compared to the wild‐type (WT) control group. Specifically, the mutant group reached an average weight of 825.0 ± 12.3 g, significantly outperforming the WT group, which reached 464.1 ± 9.2 g. Our results demonstrate that disruption of the mstnb1 gene using CRISPR/Cas9 technology induces a striking somatic weight gain rather than merely altering microscopic muscle fiber characteristics, demonstrating high biotechnological potential for shortening production time and reducing unit costs in sustainable rainbow trout aquaculture.
A. Bayır, M. Bayır, Wenjing Tao et al.· Journal of the World Aquacul...· 0 citations
The functions of noncoding variants associated with complex traits in livestock remain poorly understood. In this study, we investigated two candidate noncoding variants within the XKR4-CHCHD7 locus identified from our previous analysis. Dual-luciferase reporter assays demonstrated allele-specific regulatory activity of these two regions in bovine muscle satellite cells (BMSCs), 293T cells, and C2C12 cells. Endogenous deletion of the candidate regions using a clustered regularly interspaced short palindromic repeats (CRISPR)-based high-fidelity Cas12Max (hfCas12Max) system revealed that the region containing chr14:22840845 (SNP-0845) exerted broader effects on BMSC function including reduced proliferation and migration, altered cell-cycle progression, and enhanced myogenic differentiation. Expression screening of candidate effector genes further identified PENK and TMEM68 as downstream candidate genes for SNP-0845. Rescue experiments further showed that PENK exerted stronger recovery effects than TMEM68 on the proliferation and migration defects caused by deletion of this region, supporting PENK as a major candidate effector downstream of the SNP-0845. Functional assays showed that PENK knockdown impaired BMSC proliferation and migration while promoting myogenic differentiation, whereas PENK overexpression partially reversed these effects. In vivo Penk knockdown reduced quadriceps femoris weight and altered muscle fiber composition in mice. Collectively, our findings suggest that a noncoding regulatory region modulates BMSC fate and muscle growth-related processes through PENK.
Tianyi Wu, Feng Liu, Qunhao Niu et al.· International Journal of Mol...· 0 citations
The unresolved mechanisms underlying heterosis in beef cattle production traits hinder the optimization of crossbreeding strategies. In this study, we generated Wagyu × Hereford hybrid cattle (F1) and observed significantly enhanced growth rates compared to purebred Hereford controls, prompting further investigation into the epigenetic regulatory basis of heterosis in bovine muscle development. Combined microRNA-seq and RNA-seq profiling of the longissimus dorsi muscle identified 17 differentially expressed miRNAs and 489 differentially expressed genes (DEGs), enabling the construction of a comprehensive miRNA-mRNA regulatory network. Notably, decreased expression of bta-novel-miR-357 (novel-miR-357) and increased expression of S100A2 (S100 Calcium-Binding Protein A2) mRNA were observed in the hybrid cattle. Functional assays in primary bovine myoblasts demonstrated that novel-miR-357 and siS100A2 inhibited cell proliferation and promoted apoptosis. Dual-luciferase reporter assays confirmed that novel-miR-357 directly binds to the 3'UTR of S100A2 mRNA, establishing a targeted regulatory relationship. Furthermore, novel-miR-357 was shown to inhibit proliferation and promote apoptosis of bovine myoblasts through targeting S100A2 mRNA. These findings reveal a novel epigenetic regulatory axis (novel-miR-357/S100A2 mRNA) involved in early muscle development and provide an important molecular mechanism for deeper understanding of the epigenetic regulatory processes underlying heterosis in cattle. Nevertheless, given the polygenic complexity of heterosis, it is important to recognize that this pathway likely represents only one of several contributing factors to this complex trait.
Gaoqing Xu, Chunqi Hou, He Ding et al.· Animal Genetics· 0 citations
The phenotypic profile of a CRISPR-generated Ent2 mutant line is described and the feasibility of combining genome editing with balancer chromosome strategies in Drosophila is demonstrated, demonstrating the feasibility of combining genome editing with balancer chromosome strategies in Drosophila.
Chikun Li, Lin Li, Ying Chen et al.· Journal of Visualized Experi...· 0 citations