Animal Molecular Breeding, 2024, Vol.14, No.6, 345-353 http://animalscipublisher.com/index.php/amb 347 3 CRISPR/Cas9 Technology in Poultry Breeding 3.1 Mechanism of CRISPR/Cas9 in gene editing The CRISPR/Cas9 system is a revolutionary genome editing tool that allows for precise, cost-effective, and user-friendly modifications of genomes across various organisms, including poultry (Liu and Zhang, 2024; Wu and Li, 2024). The mechanism involves the use of a guide RNA (gRNA) to direct the Cas9 nuclease to a specific DNA sequence, where it introduces a double-strand break. This break can then be repaired by the cell's natural repair mechanisms, either through non-homologous end joining (NHEJ) or homology-directed repair (HDR) (Oishi et al., 2016). The simplicity and efficiency of this system have made it a powerful tool for targeted mutagenesis and gene knockout in chickens, as demonstrated by successful gene editing in chicken primordial germ cells (PGCs) and somatic tissues (Khwatenge and Nahashon, 2021). 3.2 Applications of CRISPR/Cas9 in poultry genetics CRISPR/Cas9 technology has been applied in various ways to advance poultry genetics. One significant application is the development of disease-resistant poultry. For instance, CRISPR/Cas9 has been used to edit viral genomes, paving the way for novel and multiplex viral vectored poultry vaccines (Vilela et al., 2020). Additionally, the technology has enabled the creation of genetically modified chickens with specific traits, such as low-allergenicity eggs and enhanced disease resistance (Chojnacka-Puchta and Sawicka, 2020). Researchers have also utilized CRISPR/Cas9 to produce transgenic progeny by targeting specific loci in chicken PGCs, demonstrating the potential for efficient genetic modification in birds (Dimitrov et al., 2016). Furthermore, the technology has been employed to study gene functions and regulatory mechanisms in chicken embryos, providing insights into developmental biology and gene expression (Gandhi et al., 2017). 3.3 Limitations and risks of CRISPR/Cas9 technology Despite its numerous advantages, CRISPR/Cas9 technology has several limitations and risks that need to be addressed. One major concern is the potential for off-target effects, where the Cas9 nuclease may introduce unintended mutations at sites other than the target locus. Although some studies have reported no detectable off-target mutations, the risk remains a significant challenge (Bai et al., 2016). Another limitation is the efficiency of HDR, which is often lower than NHEJ, making precise gene editing more difficult (Antonova et al., 2018). Additionally, the delivery of CRISPR/Cas9 components into chicken cells, particularly zygotes, poses technical challenges that need to be overcome for broader application in poultry breeding. Finally, ethical and regulatory considerations surrounding the use of gene editing in animals must be carefully navigated to ensure responsible and acceptable use of this technology (Véron et al., 2015). 4 Applications of Gene Editing in Poultry Traits Improvement 4.1 Enhancing growth and meat quality in poultry Gene editing technologies, particularly CRISPR/Cas9, have shown significant potential in enhancing growth and meat quality in poultry. By targeting specific genes associated with muscle development and growth, researchers have been able to achieve substantial improvements. For instance, the combination of CRISPR with yeast Rad52 (yRad52) has been used to enhance targeted genomic DNA editing in chicken cells, leading to increased efficiency in gene modifications. This approach has resulted in a 36.7% editing efficiency in the myostatin gene, which is known to regulate muscle growth, thereby potentially improving meat yield and quality in poultry (Wang et al., 2017). Additionally, CRISPR/Cas9 technology allows for precise modifications that can lead to the development of poultry with desirable traits, such as increased muscle mass and reduced fat content, which are critical for meat production (Khwatenge and Nahashon, 2021). 4.2 Improving disease resistance using gene editing One of the most promising applications of gene editing in poultry is the enhancement of disease resistance. CRISPR/Cas9 has been effectively used to confer resistance to viral pathogens that pose significant threats to poultry health. For example, precise editing of the NHE1 gene in chickens has rendered them resistant to the J subgroup of avian leukosis virus (ALV-J). This was achieved by introducing a single amino acid deletion in the
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