Animal Molecular Breeding 2024, Vol.14, No.2, 178-186 http://animalscipublisher.com/index.php/amb 179 editing, and the commercial prospects of this technology in dairy cattle breeding, aiming to improve animal health and productivity through innovative genetic engineering approaches. 2 CRISPR-Cas9 Technology: Mechanisms and Applications 2.1 Fundamentals of CRISPR-Cas9 gene editing CRISPR-Cas9, an acronym for Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9, is a revolutionary genome editing technology that allows for precise, targeted changes to the DNA of living organisms (Figure 1) (Zhang et al., 2021; Tao et al., 2022). Originally discovered as a part of the bacterial immune system, CRISPR-Cas9 has been adapted for use in a wide range of genetic engineering applications. The system works by utilizing a guide RNA (gRNA) to direct the Cas9 enzyme to a specific location in the genome, where it creates a double-strand break. This break can then be repaired by the cell's natural repair mechanisms, allowing for the insertion, deletion, or modification of genes (Liang et al., 2015; Liu et al., 2017; Li et al., 2021). Figure 1 The working mechanism of the CRISPR-Cas system (Adopted from Tao et al., 2022) Image caption: The bacterial defense mechanism of the CRISPR/Cas systems includes three stages: Adaptation stage: acquisition of spacer sequences; Expression stage: Generation of the crRNA and Cas protein; Interference stage: crRNA-guided nucleic acid-targeted cleavage (Adopted from Tao et al., 2022) 2.2 Applications of CRISPR-Cas9 in animal genetics CRISPR-Cas9 has been widely adopted in animal genetics for its ability to introduce precise genetic modifications. In livestock, this technology has been used to enhance disease resistance, improve productivity, and introduce desirable traits (Bevacqua et al., 2016). For instance, CRISPR-Cas9 has been employed to insert the NRAMP1 gene in cattle to confer resistance to tuberculosis and to delete the CD163 gene in pigs to make them resistant to porcine reproductive and respiratory syndrome (PRRS) (Islam et al., 2020). Beyond disease resistance, CRISPR-Cas9 is also being explored for its potential to improve other traits such as growth rate, feed efficiency, and meat quality (Barrangou and Doudna, 2016). 2.3 Advantages of CRISPR-Cas9 for disease resistance The CRISPR-Cas9 system offers several advantages for enhancing disease resistance in livestock. Firstly, it is highly specific and efficient, allowing for precise modifications with minimal off-target effects. This specificity is crucial for ensuring that only the desired genetic changes are made, reducing the risk of unintended consequences (Liang et al., 2015; Liu et al., 2023). Secondly, CRISPR-Cas9 is relatively easy to design and implement
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