International Journal of Molecular Veterinary Research 2024, Vol.14, No.1, 9-16 http://animalscipublisher.com/index.php/ijmvr 12 diversity lowers population adaptability. Irreversible mutations may occur during the gene editing of cows, potentially having long-term effects on genetic diversity. Therefore, measures need to be taken to monitor and protect the genetic diversity of the dairy cow population to prevent unexpected negative consequences. The importance of dairy cow milk fat synthesis genes in dairy product production cannot be ignored. By improving the functions of these genes, it is possible to increase the yield and quality of dairy products, meeting the growing market demand. However, current research still faces technical and practical challenges, requiring further investigation and exploration. 2 Application of CRISPR-Cas9 Technology in Agricultural Gene Editing 2.1 Development of CRISPR-Cas9 Understanding the historical development of CRISPR-Cas9 technology is crucial for a profound comprehension of its significance in agricultural gene editing. CRISPR (Clustered regularly interspaced short palindromic repeats) and Cas9 (CRISPR-associated protein 9) form a powerful duo that was initially discovered in the bacterial immune system, serving as a natural defense mechanism against viral invasions. Recognizing its potential for gene editing, researchers delved into in-depth studies of this system. The history of CRISPR technology dates back to the 1990s (Mojica et al., 1993), when researchers first observed a unique DNA sequence in bacterial genomes playing a crucial role in the bacterial immune response. However, at that time, understanding of the functionality and applications of these DNA sequences was limited. In 2012, Jennifer A. Doudna and Emmanuelle Charpentier proposed the concept of using CRISPR-Cas9 for gene editing, marking the beginning of extensive research in this field (Jinek et al., 2012). In the following years, researchers continually improved CRISPR-Cas9 technology, enhancing its accuracy and efficiency. These improvements included introducing different variants of Cas proteins, such as Cpf1, and refining the precision of editing tools. This discovery sparked widespread interest in the academic and biotechnological sectors, opening up a new avenue for scientists to precisely edit genomes. In the following years, CRISPR-Cas9 technology made significant progress and its application scope continued to expand. In the agricultural domain, CRISPR-Cas9 technology has been applied to enhance crops, endowing them with increased resistance to pests, drought, and diseases. Successful applications in other organisms have provided valuable experience and inspiration for applying CRISPR-Cas9 technology to to cow improvement, as similar principles and techniques can be applied to gene editing in different organisms, including cows (Edick et al., 2021). The historical development of CRISPR-Cas9 technology is an exhilarating journey, evolving from the accidental discovery in the bacterial immune system to a powerful gene-editing tool, bringing a huge revolution to the fields of biological science and agriculture. This progression establishes a solid foundation for the application of CRISPR-Cas9 in dairy cow improvement, providing valuable insights for future research and practical implementations. 2.2 Principles of gene editing Understanding the principles of gene editing is closely tied to comprehending the application of CRISPR-Cas9 technology in dairy cow improvement. Gene editing is a complex and precise engineering task (Horodecka and Düchler, 2021). Researchers initially select specific genes to edit, often related to milk synthesis, dairy product quality, and yield in the context of dairy cow improvement. To precisely edit target genes, researchers design guide RNA (gRNA) sequences capable of binding to specific regions of the target genes. The guide RNA directs the Cas9 protein to the vicinity of the target gene, ensuring that editing occurs at the correct location. Cas9 is an RNA-guided protein that, once bound to the guide RNA, can recognize and bind to specific sequences of the target gene. Once Cas9 binds to the target DNA, it induces a double-strand break. This marks the initiation of editing, as cells attempt to repair these breaks, usually through mechanisms like non-homologous end joining (NHEJ) or homology-directed repair (HDR). Ultimately, if the repair mechanism is successful or carefully designed by researchers, it may result in the desired genetic improvement, such as the insertion of specific gene sequences, removal of harmful gene mutations, or substitution of unfavorable genes (Figure 2).
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