The feasibility of generating MSTN/BLG double gene-edited cattle embryos using a single CRISPR/EOCas12i plasmid and SCNT is demonstrated, providing a robust platform for multiplex genome editing aimed at improving meat production and milk traits.
Abstract
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.
Gene editing in livestock using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) offers a promising approach for genetic improvement in cattle. This study evaluated germline transmission and mutation stability of double-knockout cattle generated by zygote electroporation. Previously reported myostatin/beta-lactoglobulin (MSTN/BLG) and newly generated α-1,3-galactosyltransferase (GGTA1/BLG) double-knockout cattle were produced using CRISPR/Cas9-mediated genome editing. Targeted deep sequencing demonstrated extensive somatic mosaicism across multiple tissues. Computer-assisted sperm analysis (CASA) demonstrated normal sperm motility in MSTN/BLG double-knockout males. Fertilization of wild-type oocytes produced heterozygous embryos, with mutation frequencies of 37.76 ± 10.74% at the MSTN locus and 54.80 ± 7.73% at the BLG locus, as assessed by T7 endonuclease I (T7E1) assay. MSTN/BLG double-knockout sperm were subsequently used for embryo production and for artificial insemination of GGTA1/BLG double-knockout females. Healthy offspring were successfully obtained (n = 3), alongside one stillborn calf. Targeted deep sequencing of all four progenies revealed highly variable allele frequencies that deviated substantially from the approximately 50% expected for heterozygous germline transmission. In contrast, whole-genome sequencing (WGS) results were consistent with Mendelian expectations, underscoring the limitations of PCR-based targeted sequencing for assessing germline transmission in mosaic founders. These results show that CRISPR/Cas9-edited embryos generated by electroporation can develop into healthy, sexually mature cattle capable of germline transmission. While variable transmission rates were observed owing to founder mosaicism, non-mosaic F1 offspring were successfully generated. However, direct, embryo-mediated gene-editing strategies remain technically and economically challenging for large-scale commercial calf production, and reports in cattle are limited. This study provides a reference for future applications of gene-edited embryos and their germline propagation.
Kyeong-Hyeon Eom, G. Gim, Min-Gyu Lee et al.· Theriogenology· 0 citations
ABSTRACT With the global population expected to reach 10 billion by 2050, sustainable livestock production is critical. Gene editing of the myostatin (MSTN) gene represents a promising strategy to enhance muscle growth in cattle. In this study, MSTN‐mutated founder (F0) cows were used to generate F1 offspring via ovum pick‐up, in vitro fertilization, and embryo transfer. Four F1 calves were born, all confirmed to be heterozygous for the MSTN mutation. Long‐term monitoring showed normal growth and no visible health abnormalities. Whole‐genome sequencing identified SNPs, INDELs, and structural variants, most with minimal predicted functional effects. Proteomic profiling of Longissimus dorsi muscle quantified 2947 proteins, revealing only subtle expression differences between MSTN‐mutated and wild‐type cattle. These results demonstrate stable inheritance and confirm that MSTN editing does not disrupt genome integrity or protein expression. Overall, our findings support the safety and utility of MSTN gene editing to improve livestock productivity for future food security.
G. Gim, Bae Young Choi, Jeongbin Yi et al.· Animal Genetics· 0 citations
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.
Ling Li, Ming-Xing Cao, Muhammad Farhab et al.· Functional & Integrative Gen...· 0 citations
The application of genome editing, CRISPR/Cas9 has revolutionized plant breeding by enabling precise, efficient, and targeted modification of native genes, significantly accelerating the development of improved agronomic traits of crops. Therefore, CRISPR/Cas9 technology currently the most extensively used genome editing technique worldwide because of its simple design, cost-effectiveness, high efficiency, good reproducibility, high engineering feasibility, ability to create gene knockout, RNA editing, and quick cycle. It is used to knock in or knock out genes of interest and for generating models for genetic studies. The main components of the CRISPR/Cas9 system are an RNA-guided Cas9 endonuclease and a single-guide RNA (sgRNA). The workflow of CRISPR/Cas9 gene editing comprises selecting target sites, designing and synthesizing sgRNA, introducing transformation constructs or ribonucleoprotein (RNP) in plant cells, followed by transformation and identification of edited lines. This approach bypasses the formal regulations on GMOs, thus encouraging the widespread adoption RNA-guided gene editing in agricultural sciences and biotechnology. The system is now being utilized in the biofortification of cereal crops such as rice, wheat, barley, and maize, including vegetable crops such as potato and tomato. The world's first genome-edited rice varieties are DRR Dhan 100 (Kamala) and Pusa DST Rice 1 developed by the Indian Council of Agricultural Research (ICAR), New Delhi, India in 2025 with the objective of bringing about revolutionary changes in terms of higher production, climate adaptability, and water conservation. The CRISPR/Cas9-based crop genome editing has been utilized in imparting/producing qualitative enhancement in aroma, shelf life, sweetness, and quantitative improvement in starch, protein, gamma-aminobutyric acid (GABA), oleic acid, anthocyanin, phytic acid, gluten, and steroidal glycoalkaloid contents. Some varieties have even been modified to become disease and stress-resistant. Therefore, CRISPR/Cas9 is aiding in developing climate-ready crops and improving crop quality parameters such as appearance, palatability, nutritional components, and other preferred traits. Gene editing tools are used to generate changes to the native genetic material. Unlike GMOs, which introduce novel configurations of genetic materials typically derived from other organisms, gene editing methods modify existing genetic material in ways that can yield beneficial outcomes.
Ravindra B. Malabadi, Raju K. Chalannavar· World Journal of Advanced Re...· 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 future of crop improvement using GEd technologies lies in the harmonisation or alignment of global policies and regulations to support the trade of agricultural produce and ensure that growers and consumers can benefit from GEd technology.
Michael G. K. Jones· Sugar Industry international· 0 citations