Targeted gene editing strategies in modern cattle breeding

Introduction

The rapid growth of the global population has made cattle farming increasingly important for ensuring food supply and meeting human nutritional needs. However, traditional breeding methods are slow and less efficient in improving genetic traits and production performance. Advances in biotechnology have enabled a shift from conventional breeding to molecular approaches, including gene editing. Gene editing technology is now widely used to enhance the economic value of cattle through precise genetic modifications, improving growth, productivity, and disease resistance. It also accelerates breeding by increasing the frequency of favorable alleles at quantitative trait loci (Jenko et al., 2015) [9] . Despite its potential, largescale application faces challenges such as off-target effects, animal welfare concerns, and differences in public awareness and regulatory policies across regions. Addressing these issues is essential for wider adoption. Overall, gene editing holds great promise for improving livestock productivity and meeting the rising global demand for animal protein.

Major gene editing technologies

Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas9 are the principal gene editing technologies used for precise genome modification. These tools function by creating double-strand breaks (DSBs) at specific DNA sites, which are then repaired by cellular mechanisms such as non-homologous end joining (NHEJ), often leading to insertions or deletions, or homology-directed repair (HDR), enabling precise sequence changes (Fig. 1). ZFNs are constructed by fusing zinc-finger DNA-binding motifs with the FokI endonuclease, allowing sequence-specific DNA recognition and cleavage. TALENs operate in a similar manner, combining TALE-based DNA-binding domains with the FokI nuclease, where their modular repeat structure allows flexible targeting of a wide range of DNA sequences (Carlson et al., 2012) [3] . Despite their specificity, both ZFNs and TALENs depend on protein-DNA interactions, requiring time-consuming redesign of binding domains for each target site and facing limitations in efficient delivery into cells. In contrast, the CRISPR/Cas9 system, derived from the bacterial adaptive immune system, represents a more advanced and user-friendly approach.

It utilizes a single guide RNA (sgRNA) to direct the Cas9 nuclease to specific genomic regions, enabling accurate and efficient DNA cleavage. Compared to ZFNs and TALENs, CRISPR/Cas9 is simpler to design, more cost-effective, and highly efficient, making it the most widely adopted gene editing tool (Hsu et al., 2013) [6] . It has been extensively applied in livestock improvement for enhancing traits such as growth, productivity, and disease resistance, as well as in biomedical research. For example, CRISPR/Cas9 has been used to knock out specific genes in pigs, providing a promising strategy for xenotransplantation by reducing immune rejection risks. Overall, these gene editing technologies, particularly CRISPR/Cas9, have revolutionized modern genetics and hold great potential for future advancements in animal breeding and medicine (Butler et al., 2016) [2] . Compared to conventional CRISPR/Cas9, base editors cause less genomic disruption and present lower risk, making them highly promising for applications in animal breeding research (Luo et al., 2022) [10] . Prime editors (PEs), developed in 2019, represent an advanced gene editing tool capable of generating all 12 types of point mutations as well as precise insertions and deletions without the need for double-strand breaks (DSBs) or donor DNA templates. Although their initial editing efficiency was low, continuous optimization has significantly improved their performance. Along with CRISPR/Cas9 and BEs, prime editing is now considered one of the most promising and widely used gene editing technologies due to its affordability, ease of use, high efficiency, and reduced off-target effects (Choi et al., 2022) [4] .

The CRISPR/Cas12 system has recently emerged as a powerful gene editing tool, significantly improving editing accuracy and efficiency. Compared to Cas9, Cas12 is smaller and has broader sequence recognition, and targeted amino acid modifications can further enhance its in vivo editing performance. Smaller variants such as Cas12f and Cas12n are particularly promising for gene therapy due to their compatibility with adeno-associated virus (AAV) delivery (Badon et al., 2024) [1] . In addition, a novel RNA-guided endonuclease system, termed OMEGA, has been identified, consisting of transposon-associated TnpB and IscB, which are considered evolutionary precursors of Cas12 and Cas9, respectively. These proteins are much smaller, about one-third the size of Cas12 and Cas9-making them suitable for developing miniaturized gene editing tools and single AAVbased in vivo delivery. CRISPR/Cas12 is already widely applied across biological research, while the OMEGA system holds strong potential for future breakthroughs (Wang et al., 2024) [15] . Overall, continuous advancements in gene editing technologies have led to their widespread application in cattle genetic breeding (Table 1), with significant potential to enhance livestock productivity, support sustainable agriculture, and advance medical research. Additionally, the PrP G127V variant has demonstrated strong resistance to prion diseases, and its introduction into cattle using CRISPR/Cas9 has shown successful germline transmission. However, challenges such as mosaicism still need to be addressed (Park et al., 2020) [12] . Bovine tuberculosis, caused by Mycobacterium bovis, is a chronic zoonotic disease affecting cattle productivity and human health. Gene editing strategies targeting the SP110 and NRAMP1 genes have shown promising results. Insertion of the murine SP110 gene using TALEN produced cattle resistant to infection, while overexpression or targeted insertion of NRAMP1 using CRISPR-based approaches enhanced resistance with minimal off-target effects. These modifications are heritable and demonstrate the potential of gene editing for disease control (Gao et al., 2017) [5] . Mastitis, a common and economically important disease in dairy cattle, reduces milk yield and quality. Gene editing has been used to enhance resistance by introducing antimicrobial proteins such as lysostaphin and human lysozyme into the bovine genome. These proteins are secreted in milk and exhibit strong antibacterial activity against Staphylococcus aureus. Such genetic modifications improve udder health, milk quality, and overall dairy productivity, highlighting the potential of gene editing to improve animal welfare and economic outcomes in livestock production (Jabed et al., 2012) [7] .

Improvement of milk quality

Improving milk quality through gene editing focuses on making cow's milk more similar to human breast milk to better meet human nutritional needs. This involves increasing beneficial proteins such as α-lactalbumin and lactoferrin. α-Lactalbumin is abundant in human milk (25-35%) but low in cow's milk (2-5%). Gene-edited cattle expressing recombinant human α-lactalbumin have been successfully produced using ZMI and SCNT, with milk showing similar physicochemical properties to human milk and improved nutritional value (Jackson et al., 2004) [8] . Lysozyme is another important protein with strong antimicrobial properties and a key role in human immunity. Its concentration in human milk is much higher than in cow's milk. Gene editing has been used to produce cows expressing human lysozyme, with levels and activity comparable to natural human milk. These advancements not only enhance milk's nutritional and immune benefits but also support large-scale production and improve resistance to diseases like mastitis (Piccinini et al., 2005) [13] . Milk allergy, mainly caused by β-lactoglobulin, is a significant issue in infants. Conventional processing methods are costly and may not fully eliminate allergenicity. Gene editing offers an effective alternative by reducing or eliminating β-lactoglobulin using RNAi or ZFN technologies. This results in hypoallergenic milk with improved protein composition and reduced allergenic potential without introducing foreign DNA (Saha et al., 2001) [14] . Overall, gene editing technologies enhance milk nutritional quality, improve safety by reducing allergenicity, and provide economic benefits to the dairy industry while supporting human health.

Sex control

Sex control is highly important in cattle farming, as it directly affects productivity and economic returns. Male calves are preferred in beef production due to better feed efficiency, while female calves are essential in dairy systems for increased milk production. Traditional methods such as sperm sorting, embryo sexing, and reproductive manipulation have limitations. Gene editing offers a more precise approach by targeting sex-determining mechanisms. For example, CRISPR/Cas9 has been used to insert the eGFP gene into the Y chromosome, enabling sex identification through fluorescence. The SRY gene, a key regulator of male development, has been widely studied, with knockout and knock-in strategies demonstrating its critical role in sex determination. Techniques like TALEN and HMEJ-based CRISPR approaches have successfully produced sex-reversed or targeted-sex cattle (Owen et al., 2021) [11] . Although these methods show strong potential, challenges related to efficiency and accuracy still limit their large-scale application, and further advancements are needed for commercial use.

Conclusion

Gene editing technology has emerged as a powerful tool for accelerating genetic improvement in cattle by enabling precise and efficient modification of economically important traits. Its applications in enhancing disease resistance, improving milk quality, increasing meat production, and enabling sex control highlight its transformative potential in modern livestock breeding. Despite these advancements, challenges such as off-target effects, ethical concerns, regulatory limitations, and technical constraints still need to be addressed. With continued technological improvements and supportive policies, gene editing is expected to play a crucial role in achieving sustainable livestock production, improving animal welfare, and meeting the growing global demand for high-quality animal products.

References

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