Animal Molecular Breeding 2024, Vol.14, No.2, 178-186 http://animalscipublisher.com/index.php/amb 183 6.2 Challenges and limitations in current research Despite the promising potential of CRISPR-Cas9, several challenges and limitations need to be addressed to fully realize its benefits in cattle. One major challenge is the efficiency and precision of the gene-editing process. Off-target effects, where unintended genetic modifications occur, remain a significant concern. Additionally, the delivery of CRISPR-Cas9 components into cattle cells is complex and requires further optimization to ensure high efficiency and minimal off-target activity (Zhu et al., 2020; Li et al., 2021). Another limitation is the regulatory and ethical considerations surrounding the use of gene-editing technologies in livestock. Public perception and acceptance of genetically modified animals can influence the adoption and commercialization of CRISPR-Cas9 applications in cattle breeding (Islam et al., 2020). 6.3 Strategies for enhancing precision and efficiency of gene editing To overcome the challenges associated with CRISPR-Cas9, several strategies can be employed to enhance the precision and efficiency of gene editing in cattle. One approach is the development of advanced CRISPR-Cas9 variants, such as base editors and prime editors, which allow for more precise genetic modifications with reduced off-target effects (Zhu et al., 2020; Li et al., 2021). Additionally, improving the delivery systems for CRISPR-Cas9 components, such as using viral vectors or nanoparticle-based delivery methods, can enhance the efficiency of gene editing in cattle cells (Langner et al., 2018; Chen et al., 2019). Another strategy is the use of high-throughput screening methods to identify and select the most effective guide RNAs, which can increase the specificity and success rate of CRISPR-Cas9-mediated gene editing (Kershanskaya et al., 2022). By addressing these technical challenges, the precision and efficiency of CRISPR-Cas9 in cattle can be significantly improved, paving the way for its broader application in livestock breeding programs. 7 Concluding Remarks The application of CRISPR-Cas9 technology in enhancing disease resistance in cattle has shown significant promise. CRISPR-Cas9 has emerged as a powerful tool for genome editing, enabling precise modifications in the DNA sequence to enhance disease resistance in livestock. This technology has been successfully applied to insert or delete specific genes associated with disease resistance, such as the NRAMP1 gene for tuberculosis resistance in cattle. The homology-mediated end-joining (HMEJ)-based method has been demonstrated to increase the efficiency of gene knock-ins compared to traditional homology-directed repair (HDR) methods. This has led to higher success rates in producing gene-edited cattle with predictable expression of functional genes. While the primary focus has been on cattle, CRISPR-Cas9 technology has also been applied to other livestock species, including pigs, goats, and sheep, to enhance disease resistance. This indicates the versatility and broad applicability of the technology in livestock breeding. CRISPR-Cas9 is poised to play a crucial role in the future of disease resistance in cattle. This technology can make precise genetic modifications. The decreasing cost and increasing technical ease of CRISPR-Cas9 are likely to accelerate its adoption in livestock breeding programs. This will enable the rapid development of disease-resistant cattle, reducing the reliance on traditional breeding methods that are often time-consuming and less precise. The identification of safe harbor loci, such as the ROSA26 locus, ensures that gene insertions do not disrupt other essential genes. This enhances the safety and efficiency of gene editing, making it a viable option for commercial livestock breeding. As the technology continues to advance, it is expected that CRISPR-Cas9 will become a standard tool in livestock breeding. This will not only improve disease resistance but also enhance overall animal welfare and productivity, contributing to sustainable agricultural practices. The impact of gene editing, particularly through CRISPR-Cas9, on livestock breeding is profound. By enabling precise genetic modifications, this technology offers a revolutionary approach to enhancing disease resistance in cattle and other livestock species. Disease-resistant cattle will experience fewer health issues, leading to better overall welfare and reduced need for antibiotics and other treatments. Healthier livestock translates to higher productivity and lower costs for farmers, contributing to more efficient and profitable farming operations. The ability to breed disease-resistant animals supports sustainable agricultural practices by reducing the environmental impact of livestock farming and ensuring a stable food supply. In conclusion, CRISPR-Cas9 gene editing holds
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