Animal Molecular Breeding, 2025, Vol.15, No.1, 1-8 http://animalscipublisher.com/index.php/amb 4 knock-ins. CRISPR/Cas9 has, in a relatively short period, become the dominant technology for genome editing in livestock, taking over from the earlier used ZFNs and TALENs, and has enabled precise genetic modifications for research and agricultural applications (Kalds et al., 2019; Yang, 2024). 4.2 Practical applications of CRISPR editing in goat somatic cells and embryos CRISPR/Cas9 has also been utilized to edit goat genomes successfully via both somatic cell nuclear transfer (SCNT) and direct embryo microinjection. Primary goat fetal fibroblast cells, for example, have been edited to knock in or knockout genes, and the edited cells have been used to generate live goats through SCNT (Wang et al., 2023). Cas9 mRNA and sgRNAs microinjection into zygotes has enabled effective generation of gene-edited goats, such as successful editing of MSTN and FGF5 genes, and demonstrated germline transmission of edited alleles (Wang et al., 2015; Wang et al., 2018; He et al., 2018). These techniques have been used to generate goats with enhanced production traits and altered milk composition (Li et al., 2024; Singh, 2024; Zhu et al., 2025). This technique employs the CSN2 promoter to enable the specific expression of HNP1 in the mammary gland and then convert it into an antimicrobial peptide-producing bioreactor. Our research not only confirms the feasibility of generating HNP1-expressing goats but also lays a basis for the generation of novel, high-quality dairy foods using CRISPR/Cas9 technology in goats. Moreover, it indicates the promise of CRISPR/Cas9 as a valuable tool for genetic engineering in this animal (Figure 2) (Li et al., 2024). Figure 2 Cas9-mediated HNP1 knock-in in goats at the CSN2locus (Adopted from Li et al., 2024) 4.3 Case studies of functional validation of candidate plateau adaptive genes Functional verification using CRISPR/Cas9 has targeted genes with proven or postulated roles in adaptation and production. MSTN gene knockout, for example, has resulted in goats with increased body mass and muscle fiber diameter, confirming the function of the gene in muscle growth and metabolism (He et al., 2018; Wang et al., 2018). Simultaneously, knockout and knock-in technologies have been utilized to knock in exogenous genes (e.g., fat-1, rhBChE) into specific loci and knockout endogenous genes, demonstrating the feasibility of complex genome editing in goats (Zhang et al., 2018; Wang et al., 2023). These results provide direct evidence of gene function and form the basis for plateau adaptation-related gene validation (Xuan, 2024).
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