Animal Molecular Breeding 2024, Vol.14, No.2, 178-186 http://animalscipublisher.com/index.php/amb 181 to insert the NRAMP1 gene into the bovine genome to enhance resistance to M. bovis infection. This approach involves designing single-guide RNAs (sgRNAs) to target specific loci in the bovine genome, followed by the introduction of the Cas9 nuclease to induce double-strand breaks. The NRAMP1 gene is then inserted at the target site through homologous recombination, resulting in the generation of genetically modified cattle with enhanced resistance to bTB (Islam et al., 2020). Figure 2 A working pipeline of (in vivo, in vitro, and in silico) immunogenomics for identification of disease resistance candidate gene/marker as prospective targets for genome editing (Adopted from Islam et al., 2020) Image caption: Isolation of single-cell population of a target from both phenotypic groups followed by RNA and DNA extraction separately. The RNA samples could be employed for proteomics and metabolomics profiling. On the other hand, DNA samples could also be subjected to single nucleotide polymorphisms (NSP) sequencing and genotyping, quantitative trait loci (QTL) mapping, and genome-wide association study, and epigenomics study targeting the disease resistance phenotype. Rigorous integrated bioinformatics application on all sets of omics data together enables us to identify the molecular biomarker for the target immunocompetence trait. After functional validation of the identified biomarkers in the independent population, those could be recommended as the targets for CRISPR/Cas9 mediated genome editing technology (Adopted from Islam et al., 2020) 4.4 Outcomes and implications of the case study The application of CRISPR-Cas9 technology to insert the NRAMP1 gene into the bovine genome has shown promising results in developing bTB-resistant cattle. Studies have demonstrated that the edited cattle exhibit a significant reduction in susceptibility to M. bovis infection, as evidenced by lower bacterial loads and improved immune responses compared to non-edited controls (Islam et al., 2020). This genetic approach not only enhances animal health and productivity but also has broader implications for public health by reducing the risk of zoonotic transmission of bTB. Furthermore, the success of this case study underscores the potential of CRISPR-Cas9 technology in advancing livestock disease management and improving food security.
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