Animal Molecular Breeding 2024, Vol.14, No.6 http://animalscipublisher.com/index.php/amb © 2024 AnimalSci Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved.
Animal Molecular Breeding 2024, Vol.14, No.6 http://animalscipublisher.com/index.php/amb © 2024 AnimalSci Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved. Publisher AnimalSci Publisher Editedby Editorial Team of Animal Molecular Breeding Email: edit@amb.animalscipublisher.com Website: http://animalscipublisher.com/index.php/amb Address: 11388 Stevenston Hwy, PO Box 96016, Richmond, V7A 5J5, British Columbia Canada Animal Molecular Breeding (ISSN 1927-5609) is an open access, peer reviewed journal published online by AnimalSci Publisher. The journal is publishing all the latest and outstanding research articles, letters and reviews in all areas of animal molecular breeding, containing transgenic breeding and marker assisted breeding, particularly publishing innovative research findings in the basic and applied fields of molecular genetics and novel techniques for improvement, applications of molecular enhanced products, as well as the significant evaluation of their related application field. AnimalSci Publisher is an international Open Access publisher specializing in animal molecular breeding, including genetics, breeding, as well as the related field registered at the publishing platform that is operated by Sophia Publishing Group (SPG), founded in British Columbia of Canada. All the articles published in Animal Molecular Breeding are Open Access, and are distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. AnimalSci Publisher uses CrossCheck service to identify academic plagiarism through the world’s leading plagiarism prevention tool, iParadigms, and to protect the original authors’ copyrights.
Animal Molecular Breeding (online), 2024, Vol. 14, No.6 ISSN 1927-5609 http://animalscipublisher.com/index.php/amb © 2024 AnimalSci Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved. Latest Content A Review of Gene Editing Technologies in Poultry Breeding: Focus on CRISPR/Cas9 Yanlin Wang, Qiqi Zhou, Jia Chen Animal Molecular Breeding, 2024, Vol. 14, No. 6, 345-353 The Role of TLR Genes in Canid Immunity: Insights from Wolves, Coyotes, and Dogs Jun Wang, Mengyue Chen Animal Molecular Breeding, 2024, Vol. 14, No. 6, 354-361 Molecular Markers and Genetic Variation in Water Buffalo: Insights for Conservation and Breeding JiaXuan Animal Molecular Breeding, 2024, Vol. 14, No. 6, 362-369 Effective Seasonal Breeding Strategies to Improve Goat Reproduction Rates Guoxiang Li, Liuhui Li, Chengjie Zhang Animal Molecular Breeding, 2024, Vol. 14, No. 6, 370-379 Regulation of Gene Expression in Response to Nutritional Interventions in Swine Jianli Zhong Animal Molecular Breeding, 2024, Vol. 14, No. 6, 380-387
Animal Molecular Breeding, 2024, Vol.14, No.6, 345-353 http://animalscipublisher.com/index.php/amb 345 Review Article Open Access A Review of Gene Editing Technologies in Poultry Breeding: Focus on CRISPR/Cas9 Yanlin Wang, Qiqi Zhou, Jia Chen Tropical Animal Resources Research Center, Hainan Institute of Tropical Agricultural Resources, Sanya, 572000, Hainan, China Corresponding author: jia.chen@hitar.org Animal Molecular Breeding, 2024, Vol.14, No.6 doi: 10.5376/amb.2024.14.0036 Received: 03 Nov., 2024 Accepted: 05 Dec., 2024 Published: 16 Dec., 2024 Copyright © 2024 Wang et al., This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Wang Y.L., Zhou Q.Q., and Chen J., 2024, A review of gene editing technologies in poultry breeding: focus on CRISPR/Cas9, Animal Molecular Breeding, 14(6): 345-353 (doi: 10.5376/amb.2024.14.0036) Abstract This study reviews various gene editing techniques, including CRISPR/Cas9, TALENs, and ZFNs, and emphasizes their advantages over traditional breeding. It analyzes CRISPR/Cas9 in detail, explores the use of gene editing to improve the growth, meat quality, disease resistance, and reproductive traits of poultry, reviews the ethical considerations, regulatory challenges, and safety assessments of gene edited poultry, and provides practical insights into the potential and limitations of using CRISPR/Cas9 to enhance disease resistance. Finally, it discusses future innovations in gene editing tools, integration of multi omics methods, and industry application prospects. This study aims to emphasize the transformative potential of gene editing in poultry breeding and call for ongoing research to optimize the technology and address regulatory and ethical challenges. Keywords Poultry breeding; Gene editing; CRISPR/Cas9; TALEN; ZFN 1 Introduction Poultry breeding has long been a cornerstone of agricultural practices, aimed at enhancing desirable traits such as growth rate, feed efficiency, disease resistance, and meat quality. Traditional breeding methods have relied on selective breeding and crossbreeding to achieve genetic improvements. However, these methods are often time-consuming and limited by the genetic variability present within the breeding population. The advent of molecular genetics and biotechnology has introduced new avenues for accelerating genetic gains in poultry, offering more precise and efficient tools for genetic improvement (Khwatenge and Nahashon, 2021). The emergence of gene editing technologies, particularly the CRISPR/Cas9 system, has revolutionized the field of genetic engineering in poultry. CRISPR/Cas9 allows for precise, cost-effective, and user-friendly genome editing, enabling researchers to modify gene functions, target specific genetic loci, and introduce or regulate genetic information in poultry genomes (Vilela et al., 2020). This technology has been successfully applied to enhance disease resistance, such as rendering chickens resistant to avian leukosis virus subgroup J through targeted gene deletions (Koslová et al., 2020). Additionally, CRISPR/Cas9 has been utilized to develop recombinant vaccines and improve host-virus interactions, showcasing its potential in advancing poultry health and productivity. This study explores the progress of applying CRISPR/Cas9 to poultry species, evaluates its potential benefits and limitations, discusses its impact on the poultry industry, and analyzes the current status and future prospects of gene editing technology in poultry breeding. This study aims to emphasize the transformative impact of CRISPR/Cas9 on poultry farming and its role in addressing industry challenges. 2 Gene Editing Technologies in Poultry Breeding 2.1 Overview of gene editing tools (CRISPR/Cas9, TALEN, ZFN) Gene editing technologies have revolutionized the field of poultry breeding, offering precise and efficient methods to modify the avian genome. The most prominent tools include CRISPR/Cas9, TALEN (Transcription Activator-Like Effector Nucleases), and ZFN (Zinc Finger Nucleases) (Gupta et al., 2019). Among these, CRISPR/Cas9 stands out due to its simplicity, cost-effectiveness, and versatility. CRISPR/Cas9 allows for targeted gene modifications by utilizing a guide RNA to direct the Cas9 nuclease to specific DNA sequences, enabling
Animal Molecular Breeding, 2024, Vol.14, No.6, 345-353 http://animalscipublisher.com/index.php/amb 346 precise cuts and subsequent gene editing (Khwatenge and Nahashon, 2021). TALEN and ZFN, while also effective, are more complex and less user-friendly compared to CRISPR/Cas9. TALENs use engineered proteins to bind and cut specific DNA sequences, whereas ZFNs employ zinc finger domains to achieve similar outcomes. Despite their differences, all three technologies have been successfully applied in various organisms, including poultry, to achieve targeted genetic modifications (Figure 1) (Islam et al., 2020). Figure 1 Nuclease-based genome editors (Adopted from Islam et al., 2020) Image caption: (A). Zinc Finger Nuclease (B). Transcription-Activator Like Effector Nuclease (TALEN). (C). Schematic diagram showing genome editing using CRISPR/Cas9 system. The Cas9 induces DNA double-strand break (DSB) which are repaired either by imperfect nonhomologous end-joining (NHEJ) to generate insertion or deletion (indels) or if a repair is provided, by homology-directed repair (HDR) (Adopted from Islam et al., 2020) 2.2 Advantages of gene editing over traditional breeding methods Gene editing technologies offer several advantages over traditional breeding methods. Traditional breeding relies on selective mating and phenotypic selection, which can be time-consuming and less precise. In contrast, gene editing allows for the direct modification of specific genes, leading to faster and more accurate results. For instance, CRISPR/Cas9 has been used to introduce desirable traits in poultry, such as disease resistance and improved production characteristics, which would be challenging to achieve through conventional breeding (Vilela et al., 2020). Additionally, gene editing can overcome the limitations of genetic diversity in breeding populations by introducing new genetic variations that are not present in the existing gene pool. This capability is particularly beneficial in enhancing traits that are difficult to improve through traditional methods, such as resistance to avian diseases and improved feed efficiency. 2.3 Challenges in applying gene editing in poultry Despite its potential, the application of gene editing in poultry breeding faces several challenges. One significant hurdle is the difficulty in accessing and manipulating poultry zygotes, which complicates the gene editing process (Oishi et al., 2016). Additionally, there are technical challenges related to the efficiency and specificity of gene editing tools. For example, off-target effects, where unintended genetic modifications occur, remain a concern with CRISPR/Cas9 technology (Zhang et al., 2019). Furthermore, ethical and regulatory issues surrounding the use of gene editing in animals pose additional challenges. There is ongoing debate about the acceptability and safety of genetically modified organisms (GMOs), which can impact the adoption of these technologies in the poultry industry. Addressing these challenges requires continued research and development to improve the precision and efficiency of gene editing tools, as well as efforts to engage with regulatory bodies and the public to ensure the responsible use of these technologies in poultry breeding.
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
Animal Molecular Breeding, 2024, Vol.14, No.6, 345-353 http://animalscipublisher.com/index.php/amb 348 gene encoding the receptor required for ALV-J infection, resulting in chickens that are resistant to this virus without any visible side effects (Koslová et al., 2020). This demonstrates the potential of CRISPR/Cas9 to create poultry breeds that are less susceptible to diseases, thereby reducing the need for antibiotics and improving overall flock health (Wang et al., 2022). 4.3 Modifying reproductive traits for better efficiency Gene editing also holds promise for improving reproductive traits in poultry, which can lead to better breeding efficiency and productivity. By targeting genes involved in reproductive processes, researchers can enhance traits such as egg production, fertility, and hatchability. CRISPR/Cas9 technology has been used to modify the genomes of poultry to better understand and manipulate reproductive traits. For instance, the development of innovative genome-edited avian models, including specific chicken bioreactors and knock-in/out chickens, has facilitated the study and improvement of reproductive efficiency (Chojnacka-Puchta and Sawicka, 2020). These advancements can lead to more efficient breeding programs and higher productivity in the poultry industry. 5 Ethical, Regulatory, and Safety Considerations 5.1 Ethical concerns surrounding gene editing in poultry The application of CRISPR/Cas9 in poultry breeding raises significant ethical concerns. One of the primary issues is the potential for unforeseen and undesirable effects, which could impact animal welfare and biodiversity. Ethical debates often focus on the morality of altering the genetic makeup of living organisms, particularly when it involves germline modifications that can be passed on to future generations (Zhang et al., 2020). Additionally, there is concern about the potential exploitation of this technology for eugenics or other morally contentious purposes (Shinwari et al., 2018). The ease and precision of CRISPR/Cas9 make it a powerful tool, but this also means that its misuse could have far-reaching consequences, necessitating stringent ethical guidelines and public discourse to ensure responsible use (Véron et al., 2015). 5.2 Regulatory landscape for gene editing technologies The regulatory landscape for gene editing technologies like CRISPR/Cas9 is complex and varies significantly across different regions. Many countries are still in the process of developing comprehensive regulatory frameworks to address the unique challenges posed by these technologies. For instance, the non-traceability of modifications and the blurring of boundaries between natural and genetically modified organisms call for a rethinking of existing regulatory approaches (Bartkowski et al., 2018). International standards and guidelines are crucial to harmonize regulations and ensure the safe and ethical application of gene editing. Organizations such as the National Academies and the Biological Toxins and Weapons Convention (BTWC) are working towards establishing these standards, but more engaged international dialogue is needed to address the rapid advancements in this field (DiEuliis and Giordano, 2017). 5.3 Safety and risk assessment of gene-edited poultry products Safety and risk assessment are critical components of the regulatory process for gene-edited poultry products. The primary concern is the potential for off-target effects, which could lead to unintended genetic changes with unknown consequences (Dimitrov et al., 2016; Memi et al., 2018). Additionally, the long-term impacts on animal health and the environment need to be thoroughly evaluated. Studies have shown that precise CRISPR/Cas9 editing can confer resistance to diseases such as avian leukosis virus without visible side effects, indicating the potential for safe application (Koslová et al., 2020). However, continuous monitoring and periodic assessment are essential to ensure that any risks are identified and mitigated promptly. The involvement of biosafety and biosecurity communities in these assessments is also crucial to address the dual-use potential of gene editing technologies. 6 Case Study: Application of CRISPR/Cas9 in Poultry Breeding 6.1 Overview of the case study and selected poultry species This case study focuses on the application of CRISPR/Cas9 technology in chickens, specifically targeting the avian leukosis virus subgroup J (ALV-J). ALV-J is a significant pathogen in poultry, causing economic losses due
Animal Molecular Breeding, 2024, Vol.14, No.6, 345-353 http://animalscipublisher.com/index.php/amb 349 to its impact on chicken health and productivity. The study selected chickens as the model species due to their importance in the poultry industry and the feasibility of genetic modifications in this species (Oishi et al., 2016). 6.2 Implementation of CRISPR/Cas9 for disease resistance The implementation of CRISPR/Cas9 in this case involved precise editing of the chicken Na+/H+ exchanger type 1 (chNHE1) gene, which encodes a receptor critical for ALV-J entry into chicken cells. Researchers introduced a single amino acid deletion at tryptophan residue number 38 (W38) in the chNHE1 gene. This specific mutation was chosen because W38 is essential for the virus's ability to infect chicken cells (Figure 2) (Koslová et al., 2020). Figure 2 Design of guide RNA and homologous recombination ssODN for CRISPR/Cas9 gene editing of chicken ALV-J receptor chNHE1in primordial germ cells (Adopted from Koslová et al., 2020) Image caption: (A) The structure of coding exons and introns of chNHE1(Top), the CRISPR/Cas9 target sequence of exon 1 with the guide RNA (gRNA) complementary sequence (underlined) and the TGG triplet encoding W38 (red on the yellow background; Middle), and the central part of the ssODN template for homologous recombination with deleted TGG triplet and a single nucleotide substitution (in red) creating the BsaI restriction site (Bottom). (B) Preparation of ΔW38 chickens: schematic representation of the workflow and timeline (Adopted from Koslová et al., 2020) The CRISPR/Cas9 system was used to delete the W38 residue in chicken primordial germ cells (PGCs). These edited PGCs were then used to produce gene-edited chickens. The resulting ΔW38 homozygous chickens were tested for resistance to ALV-J both in vitro and in vivo. The results showed that ΔW38 homozygous chickens were resistant to ALV-J, whereas ΔW38 heterozygotes and wild-type birds remained susceptible (Table 1) (Koslová et al., 2020). Table 1 ALV-J viremia in chickens inoculated with RCASBP(J)GFP (Adopted from Koslová et al., 2020) chNHE1 genotype Chicken no. Age of chicken, d Virus titer, IU/mL 6dp.i. 13dp.i. W38−/− 604 28 0 0 W38−/− 606 28 0 0 W38−/− 611 20 0 0 W38−/− 612 20 0 0 W38−/− 615 14 0 0 W38+/− 601 28 0 101 W38+/− 603 28 0 102 W38+/− 605 28 0 101 W38+/− 610 20 102 103 W38+/− 616 20 0 103 W38+/− 618 14 0 102 W38+/− 619 14 101 103 W38+/+ 617 14 102 103
Animal Molecular Breeding, 2024, Vol.14, No.6, 345-353 http://animalscipublisher.com/index.php/amb 350 6.3 Analysis of results and implications for future breeding The results of this study demonstrated that precise CRISPR/Cas9 gene editing could confer resistance to ALV-J in chickens without any visible side effects. This finding is significant as it highlights the potential of CRISPR/Cas9 technology to enhance disease resistance in poultry, thereby reducing economic losses and improving animal welfare (Islam et al., 2020; Gul et al., 2022). The successful implementation of CRISPR/Cas9 in this case study opens up new possibilities for breeding disease-resistant poultry. By targeting specific genes associated with disease susceptibility, it is possible to develop chicken strains that are resilient to various pathogens. This approach could significantly decrease the reliance on antibiotics and vaccines, promoting more sustainable and health-conscious poultry farming practices (Vilela et al., 2020). 7 Future Directions in Gene Editing for Poultry Breeding 7.1 Innovations in gene editing tools and techniques The CRISPR/Cas9 system has revolutionized gene editing in poultry, allowing for precise modifications in the avian genome. Recent advancements have focused on improving the efficiency and specificity of these tools. For instance, CRISPR technology has been successfully applied to modify genes in chickens and quails, enhancing genetic variations that are beneficial for poultry production. However, there is still room for improvement in the techniques used to achieve heritable edited traits in birds, which are currently quite involved and specific to avian reproductive biology (Tizard et al., 2019). Future innovations may include more refined methods for germline editing and the development of new gene-editing tools that can overcome the current limitations of CRISPR/Cas9 (Preethi et al., 2020). 7.2 Potential for integrating multi-omics approaches The integration of multi-omics approaches, including genomics, transcriptomics, proteomics, and metabolomics, holds significant potential for advancing poultry breeding. These technologies can provide comprehensive insights into the genetic and molecular basis of important traits such as disease resistance, productivity, and welfare (Dehau et al., 2022). The decreasing cost of omics technologies makes their implementation in routine poultry monitoring systems more feasible, potentially leading to the development of diagnostic tests based on disease-specific biomarkers (Goossens et al., 2022). By combining multi-omics data with gene editing, researchers can achieve a more precise and holistic understanding of complex traits, thereby enhancing the effectiveness of breeding programs (Langridge and Fleury, 2011; Mahmood et al., 2022). 7.3 Prospects for commercialization and industry adoption The commercialization and industry adoption of gene editing technologies in poultry breeding face several challenges, including regulatory hurdles and public perception. The regulatory landscape for genome-edited animals varies significantly across countries, which could lead to disparities in the adoption of these technologies and potential trade disruptions (Bishop and Eenennaam, 2020). Despite these challenges, the potential benefits of gene editing, such as improved disease resistance and enhanced productivity, make it an attractive option for the poultry industry. The successful commercialization of gene-edited poultry will likely depend on achieving regulatory harmony and addressing public concerns about the safety and ethics of these technologies. As the technology matures and becomes more widely accepted, it is expected that gene editing will play a crucial role in the future of poultry breeding (Khwatenge and Nahashon, 2021). 8 Concluding Remarks The application of CRISPR/Cas9 technology in poultry breeding has shown significant promise and progress. CRISPR/Cas9 has been successfully utilized to modify gene functions for various purposes, including transcriptional regulation, gene targeting, and epigenetic modification in poultry species, particularly chickens and quails. This technology has enabled the precise editing of viral genomes, aiding in the development of novel poultry vaccines and enhancing resistance to avian diseases. Additionally, CRISPR/Cas9 has been employed to achieve targeted mutagenesis and homologous recombination in chicken cell lines, demonstrating high efficiency
Animal Molecular Breeding, 2024, Vol.14, No.6, 345-353 http://animalscipublisher.com/index.php/amb 351 and specificity. The optimization of CRISPR/Cas9 for early chick embryos has further improved the efficiency and specificity of gene knockouts, facilitating advanced genetic studies in poultry. Moreover, innovative delivery systems, such as using Marek’s disease virus, have been explored to enhance the practical application of CRISPR/Cas9 in poultry. Future research should focus on addressing the limitations of CRISPR/Cas9 technology in poultry breeding. One critical area is improving the efficiency and precision of gene editing to minimize off-target effects and ensure stable genetic modifications. Developing more robust delivery systems for CRISPR/Cas9 components, such as gesicle technology, could enhance the practical application of this technology in poultry. Additionally, exploring the use of tissue-specific promoters and spatiotemporal control strategies could further refine gene editing outcomes and reduce unintended consequences. Research should also investigate the long-term effects of CRISPR/Cas9-mediated genetic modifications on poultry health and productivity to ensure the safety and sustainability of this technology in the poultry industry. The integration of CRISPR/Cas9 technology into poultry breeding holds transformative potential for the industry. By enabling precise genetic modifications, this technology can enhance disease resistance, improve production traits, and contribute to the development of innovative poultry vaccines. The advancements in CRISPR/Cas9 applications in poultry not only promise to improve the efficiency and sustainability of poultry production but also open new avenues for scientific research and biotechnological innovations. As research continues to address the current limitations and optimize the use of CRISPR/Cas9, the full potential of gene editing in poultry breeding will likely be realized, leading to significant advancements in the field. Acknowledgments We are grateful to Mrs. Yuan for critically reading the manuscript and providing valuable feedback that improved the clarity of the text. Conflict of Interest Disclosure The authors affirm that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest. References Antonova E., Glazova O., Gaponova A., Eremyan A., Zvereva S., Grebenkina N., Volkova N., and Volchkov P., 2018, Successful CRISPR/Cas9 mediated homologous recombination in a chicken cell line, F1000Research, 7: 238. Bai Y., He L., Li P., Xu K., Shao S., Ren C., Liu Z., Wei Z., and Zhang Z., 2016, Efficient genome editing in chicken DF-1 cells using the CRISPR/Cas9 system, G3: Genes|Genomes|Genetics, 6: 917-923. https://doi.org/10.1534/g3.116.027706 Bartkowski B., Theesfeld I., Pirscher F., and Timaeus J., 2018, Snipping around for food: Economic, ethical and policy implications of CRISPR/Cas genome editing, Geoforum, 96: 172-180. https://doi.org/10.1016/J.GEOFORUM.2018.07.017 Bishop T., and Eenennaam A., 2020, Genome editing approaches to augment livestock breeding programs, Journal of Experimental Biology, 223(Suppl_1): jeb207159. https://doi.org/10.1242/jeb.207159 Chojnacka-Puchta L., and Sawicka D., 2020, CRISPR/Cas9 gene editing in a chicken model: current approaches and applications, Journal of Applied Genetics, 61: 221-229. https://doi.org/10.1007/s13353-020-00537-9 Dehau T., Ducatelle R., Immerseel F., and Goossens E., 2022, Omics technologies in poultry health and productivity-part 1: current use in poultry research, Avian Pathology, 51: 407-417. https://doi.org/10.1080/03079457.2022.2086447 DiEuliis D., and Giordano J., 2017, Gene editing using CRISPR/Cas9: implications for dual-use and biosecurity, Protein and Cell, 9: 239-240. https://doi.org/10.1007/s13238-017-0493-4 Dimitrov L., Pedersen D., Ching K., Yi H., Collarini E., Izquierdo S., Lavoir M., and Leighton P., 2016, Germline gene editing in chickens by efficient CRISPR-Mediated homologous recombination in primordial germ cells, PLoS One, 11(4): e0154303. https://doi.org/10.1371/journal.pone.0154303
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Animal Molecular Breeding, 2024, Vol.14, No.6, 345-353 http://animalscipublisher.com/index.php/amb 353 Zhang D., Hussain A., Manghwar H., Xie K., Xie S., Zhao S., Larkin R., Qing P., Jin S., and Ding F., 2020, Genome editing with the CRISPR‐Cas system: an art, ethics and global regulatory perspective, Plant Biotechnology Journal, 18: 1651-1669. https://doi.org/10.1111/pbi.13383 Zhang J., Liu J., Yang W., Cui M., Dai B., Dong Y., Yang J., Zhang X., Liu D., Liang H., and Cang M., 2019, Comparison of gene editing efficiencies of CRISPR/Cas9 and TALEN for generation of MSTN knock-out cashmere goats, Theriogenology, 132: 1-11. https://doi.org/10.1016/j.theriogenology.2019.03.029 Disclaimer/Publisher’s Note The statements, opinions, and data contained in all publications are solely those of the individual authors and contributors and do not represent the views of the publishing house and/or its editors. The publisher and/or its editors disclaim all responsibility for any harm or damage to persons or property that may result from the application of ideas, methods, instructions, or products discussed in the content. Publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Animal Molecular Breeding, 2024, Vol.14, No.6, 354-361 http://animalscipublisher.com/index.php/amb 354 Research Insight Open Access TheRole of TLRGenes in Canid Immunity: Insights from Wolves, Coyotes, and Dogs Jun Wang, Mengyue Chen Animal Science Research Center, Cuixi Academy of Biotechnology, Zhuji, 311800, Zhejiang, China Corresponding author: mengyue.chen@cuixi.org Animal Molecular Breeding, 2024, Vol.14, No.6 doi: 10.5376/amb.2024.14.0037 Received: 05 Nov., 2024 Accepted: 08 Dec., 2024 Published: 20 Dec., 2024 Copyright © 2024 Wang and Chen, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Wang J., and Chen M.Y., 2024, The role of TLRgenes in canid immunity: insights from wolves, coyotes, and dogs, Animal Molecular Breeding, 14(6): 354-361 (doi: 10.5376/amb.2024.14.0037) Abstract This study systematically reviews the critical role of Toll-like receptor (TLR) genes in canid immunity, focusing on the genetic polymorphisms, functional mechanisms, and evolutionary dynamics of TLRgenes in wolves, coyotes, and domestic dogs. As an essential component of the innate immune system, TLRs recognize pathogen-associated molecular patterns (PAMPs) and activate immune signaling pathways, playing a central role in combating bacterial, viral, and fungal pathogens. The diversity of TLRgenes is closely linked to host immune competence and disease susceptibility, with African wild dogs exhibiting higher TLR polymorphism, potentially enhancing their resistance to canine distemper virus (CDV). This study reveals how specific variations in genes such as TLR2, TLR4, and TLR7 influence immune responses and highlights the importance of pathogen-mediated selection pressures in maintaining genetic diversity. It further explores the potential applications of TLRgenes in conservation and breeding, including the use of genetic markers for marker-assisted selection to enhance disease resistance in domestic dogs and the management of genetic diversity to address regional pathogen pressures in wild canids. By synthesizing current research findings, this study identifies future research directions, emphasizing the application of genomic and transcriptomic technologies in elucidating the functions and evolution of TLRgenes. Keywords Toll-like receptor (TLR); Canid immunity; Genetic diversity; Pathogen resistance; Conservation and breeding 1 Introduction Canid species, including wolves, coyotes, and domestic dogs, possess a complex and highly adaptive immune system that enables them to combat a wide array of pathogens. The immune system is a complex network of cells, tissues, and organs that work together to defend the body against harmful pathogens and maintain homeostasis (Liu and Huang, 2024). The innate immune system, which serves as the first line of defense, plays a crucial role in recognizing and responding to infectious agents. Among the key components of this system are Toll-like receptors (TLRs), which are essential for detecting microbial infections and initiating immune responses. Understanding the genetic and functional diversity of TLRs in canids is vital for comprehending their immune capabilities and disease susceptibilities. Toll-like receptors (TLRs) are a family of pattern recognition receptors that identify pathogen-associated molecular patterns (PAMPs) and activate downstream signaling pathways to elicit immune responses. TLRs are pivotal in recognizing a broad spectrum of pathogens, including viruses, bacteria, and fungi. Polymorphisms in TLR genes can significantly influence the effectiveness of immune responses and the susceptibility of hosts to various diseases. For instance, variations in TLR2, TLR3, TLR4, TLR7, and TLR8 have been linked to differential susceptibility to canine distemper virus (CDV) in African wild dogs and lions, highlighting the critical role of TLR diversity in disease resistance (Loots et al., 2018). Additionally, TLR polymorphisms have been shown to undergo pathogen-mediated selection, maintaining genetic diversity in natural populations and influencing disease susceptibility (Quéméré et al., 2021). This study summarizes the genetic variations in TLR genes across different canid species, explores the evolutionary pressures shaping TLR diversity in wild populations, evaluates the functional impacts of TLR polymorphisms on immune responses to specific pathogens, and highlights the significance of TLR research in
Animal Molecular Breeding, 2024, Vol.14, No.6, 354-361 http://animalscipublisher.com/index.php/amb 355 canid conservation and disease management. It aims to provide comprehensive insights into how these receptors contribute to disease resistance and overall immune competence. 2TLRGenes: Structure and Function 2.1 General overview of TLRgene families Toll-like receptors (TLRs) are a critical component of the innate immune system, serving as pattern recognition receptors (PRRs) that detect pathogen-associated molecular patterns (PAMPs) from various microbes. TLRs are evolutionarily conserved across species, including canids such as wolves, coyotes, and dogs. These receptors are integral in recognizing a wide array of microbial components, including bacterial lipoproteins, lipopolysaccharides, and viral nucleic acids, thereby initiating immune responses (Dolasia et al., 2018; Fitzgerald and Kagan, 2020; Guo et al., 2023). The TLR family is composed of multiple members, each with specific ligand recognition capabilities, such as TLR1, TLR2, TLR4, and TLR5, which are known to recognize bacterial components, and TLR3, TLR7, TLR8, and TLR9, which are involved in viral recognition (Mukherjee et al., 2019; Zhou et al., 2021). 2.2 Mechanisms of action in immune response Upon recognition of PAMPs, TLRs activate downstream signaling pathways that lead to the production of cytokines and other inflammatory mediators. This process is primarily mediated through adaptor proteins such as MyD88 (myeloid differentiation primary-response protein 88), which forms a complex known as the myddosome. This complex recruits and activates IL-1R-associated kinases (IRAKs), which are crucial for the propagation of the signal leading to the activation of transcription factors like NF-κB and the production of pro-inflammatory cytokines (Guo et al., 2023; Pereira and Gazzinelli, 2023). Additionally, TLRs can form supramolecular organizing centers (SMOCs) that ensure precise and robust signaling responses (Fitzgerald and Kagan, 2020). The activation of TLRs not only triggers immediate innate immune responses but also shapes adaptive immunity by influencing the activation and differentiation of T and B cells (Dolasia et al., 2018). 2.3 Evolutionary perspectives of TLRgenes in canids The evolutionary dynamics of TLR genes in canids reveal significant polymorphism and adaptive evolution, driven by pathogen-mediated selection. Studies have shown that TLRgenes exhibit high levels of genetic diversity, similar to the major histocompatibility complex (MHC) genes, which are crucial for adaptive immunity (Těšický et al., 2020; Quéméré et al., 2021). This diversity is maintained through balancing selection, where different alleles confer advantages against various pathogens, thus promoting heterozygosity within populations. For instance, specific TLR polymorphisms have been associated with differential susceptibility to infections, highlighting their role in the evolutionary arms race between hosts and pathogens (Mukherjee et al., 2019; Wang et al., 2021). In canids, the conservation and variation of TLRgenes suggest a complex interplay between genetic drift, selection pressures, and environmental factors, contributing to the robustness of their immune responses (Těšický et al., 2020; Guo et al., 2023). 3 Comparative Insights Across Canid Species 3.1 TLRgene variability in wolves In North American gray wolves, the TLR3 gene has been studied in relation to the KB allele, which is associated with black coat color. This allele, introduced through hybridization with dogs, underwent a selective sweep, increasing its frequency in wild wolf populations. Despite this positive selection, wolves with the KBB genotype exhibit lower fitness compared to those with the KyB genotype, suggesting pleiotropic effects of the KB allele on phenotypes beyond coat color. However, studies have shown that the K locus genotype does not predict the transcriptional response to TLR3 pathway stimulation or infection by canine distemper virus (CDV), indicating that the gene expression response does not explain the pleiotropic effects on fitness (Figure 1) (Johnston et al., 2021).
Animal Molecular Breeding, 2024, Vol.14, No.6, 354-361 http://animalscipublisher.com/index.php/amb 356 Figure 1CBD103gene expression in North American gray wolves (Adopted from Johnston et al., 2021) Image caption: (A) Coat color polymorphism in North American gray wolves in Yellowstone National Park, in which coat color can be gray or black (photo credit Dan Stahler/National Park Service photo). Black coat color is dominantly inherited, conferred by a 3 base pair coding deletion in CBD103. (B) CBD103 expression, relative to expression in dog testis, across tissues of a recently deceased pregnant female Kyy wolf. Error bars represent standard errors across RT-qPCR replicates (2–3 replicates per tissue). A single tissue sample was collected for each tissue except fetus and placenta, which each represent 2 tissue samples (i.e., from 2 fetuses). (C) Absolute expression of annotated beta defensins in epidermal keratinocytes (N = 23), fibroblasts (N = 6), and whole blood (N= 25) from North American gray wolves. Only CBD103 is highly expressed in keratinocytes (Adopted from Johnston et al., 2021) 3.2 Functional implications of TLRgenes in coyotes While specific studies on TLRgenes in coyotes are limited, insights can be drawn from related species and general TLR functionality. TLRs play a crucial role in recognizing pathogens and initiating immune responses. For instance, in other wild species, TLR genes exhibit significant polymorphism, which is maintained by pathogen-mediated selection. This polymorphism influences disease susceptibility and immune responses, as seen in studies on roe deer where TLR2 polymorphism is shaped by antagonistic selection pressures from different pathogens (Quéméré et al., 2021). Similar mechanisms are likely at play in coyotes, where TLR gene variability could influence their ability to respond to diverse pathogen challenges in their environment. 3.3 TLRgene adaptations in domestic dogs Domestic dogs have undergone significant genetic adaptations during domestication, including changes in TLR genes. Whole-genome sequencing of African dogs has revealed positive selection in genes linked to immunity, such as ADGRE1, which provides protective host defense against Plasmodium infections. This gene is also associated with severe malaria resistance in humans, highlighting the role of TLR-related genes in adapting to tropical environments (Liu et al., 2018). Additionally, structural variations in the dog genome, including insertions and deletions, have been linked to immune system functions, further illustrating the impact of domestication on TLRgene adaptations (Wang et al., 2018). 4 TLRGenes and Disease Resistance in Canids 4.1 Role of TLRs in bacterial and viral infections Toll-like receptors (TLRs) are crucial components of the innate immune system, recognizing pathogen-associated molecular patterns (PAMPs) and initiating immune responses against a variety of pathogens, including bacteria and viruses. TLRs such as TLR2, TLR4, and TLR7 have been shown to play significant roles in the immune
Animal Molecular Breeding, 2024, Vol.14, No.6, 354-361 http://animalscipublisher.com/index.php/amb 357 response to infections in canids. For instance, TLR2 is involved in recognizing bacterial lipoproteins, while TLR4 detects lipopolysaccharides from Gram-negative bacteria, and TLR7 is essential for recognizing viral single-stranded RNA (Loots et al., 2018; Mukherjee et al., 2019; Heni et al., 2020). Polymorphisms in these TLR genes can influence the host's susceptibility to infections. For example, a study on African wild dogs and lions revealed that TLR2 polymorphisms might affect susceptibility to canine distemper virus (CDV), with specific amino acid changes potentially altering TLR2 function and expression (Loots et al., 2018). 4.2 Case studies: disease susceptibility linked to TLR variants Several case studies have highlighted the link between TLRgene variants and disease susceptibility in canids. In a study of CDV outbreaks in South Africa, non-synonymous single nucleotide polymorphisms (SNPs) in TLR2, TLR3, TLR4, TLR7, and TLR8 were investigated. The study found a higher rate of TLR polymorphisms in African wild dogs compared to lions, with a specific TLR2 variant (Met527Thr) potentially influencing CDV susceptibility in lions (Loots et al., 2018). Another study on roe deer demonstrated that TLR2 polymorphisms are subject to pathogen-mediated selection, affecting susceptibility to infections like Toxoplasma and Chlamydia (Quéméré et al., 2021). These findings underscore the importance of TLR genetic diversity in disease resistance and susceptibility in wildlife. 4.3 Genetic markers and breeding for enhanced immunity The identification of TLR polymorphisms as genetic markers offers promising avenues for breeding programs aimed at enhancing disease resistance in canids. For example, the presence of specific TLR variants can be used to select individuals with enhanced immune responses for breeding. In dairy cattle, TLR SNPs have been identified as potential markers for breeding strategies to improve resistance to diseases such as mastitis and bovine tuberculosis (Maljković et al., 2023). Similarly, in canids, TLR polymorphisms could be utilized to develop marker-assisted selection programs to enhance disease resistance. The use of retrotransposon insertion polymorphisms (RIPs) in TLRgenes, such as the 192 bp ERV insertion in TLR6, has shown potential in increasing TLR expression and downstream immune responses, which could be applied in breeding programs for disease-resistant animals (Wang et al., 2021). 5 Case Study 5.1 Regional pathogen pressure in wild and domestic canids Pathogen pressure varies significantly across regions and species, influencing the genetic diversity of Toll-like receptors (TLRs) in canids. For instance, in South Africa, outbreaks of canine distemper virus (CDV) in lions and African wild dogs revealed different susceptibilities and TLR polymorphisms. Lions exhibited lower TLR diversity compared to African wild dogs, which showed higher rates of polymorphism within TLR loci (Loots et al., 2018). Similarly, in a study on neotropical rodents, TLR4 haplotypes varied across landscapes with different degrees of anthropogenic disturbance, affecting resistance to gastrointestinal nematodes and Hepacivirus (Heni et al., 2020). These findings underscore the importance of regional pathogen pressures in shaping TLR diversity and pathogen resistance in wild and domestic canids. 5.2 Sampling and genotyping of TLR variants Sampling and genotyping of TLR variants involve collecting tissue or blood samples from canids and analyzing the genetic sequences of TLR genes. In the study of CDV outbreaks in South Africa, researchers investigated non-synonymous single nucleotide polymorphisms (SNPs) in the coding regions of TLR2, TLR3, TLR4, TLR7, and TLR8 genes (Loots et al., 2018). Similarly, in a study on porcine TLR genes, bioinformatic prediction combined with PCR-based amplification was used to screen for retrotransposon insertion polymorphisms (RIPs) (Wang et al., 2021). These methods allow for the identification of specific TLR variants and their association with disease susceptibility. 5.3 Correlation between TLR variants and pathogen resistance The correlation between TLR variants and pathogen resistance is evident in several studies. For example, a single amino acid change (Met527Thr) within the leucine-rich repeat of TLR2 was observed in a surviving lioness
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