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Kim, Y.; Woo, S.; Han, J.Y. Applications of Genome Editing for Avian Model Development. Encyclopedia. Available online: https://encyclopedia.pub/entry/43697 (accessed on 09 July 2025).
Kim Y, Woo S, Han JY. Applications of Genome Editing for Avian Model Development. Encyclopedia. Available at: https://encyclopedia.pub/entry/43697. Accessed July 09, 2025.
Kim, Young-Min, Seung-Je Woo, Jae Yong Han. "Applications of Genome Editing for Avian Model Development" Encyclopedia, https://encyclopedia.pub/entry/43697 (accessed July 09, 2025).
Kim, Y., Woo, S., & Han, J.Y. (2023, May 03). Applications of Genome Editing for Avian Model Development. In Encyclopedia. https://encyclopedia.pub/entry/43697
Kim, Young-Min, et al. "Applications of Genome Editing for Avian Model Development." Encyclopedia. Web. 03 May, 2023.
Applications of Genome Editing for Avian Model Development
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This entry is adapted from the peer-reviewed paper 10.3390/genes14040899

Avian models are valuable for studies of development and reproduction and have important implications for food production. Rapid advances in genome-editing technologies have enabled the establishment of avian species as unique agricultural, industrial, disease-resistant, and pharmaceutical models. The direct introduction of genome-editing tools, such as the clustered regularly interspaced short palindromic repeats (CRISPR) system, into early embryos has been achieved in various animal taxa. The advancement of avian germline transmission and genome editing technology enabled researchers to develop various genome edited avian models, including a disease resistant model, efficient bioreactor, and academic model for scientific use.

avian model genome editing primordial germ cell (PGC)

1. Genome Editing for the Development of Disease-Resistant Avian Models

Disease control and prevention in birds is an essential prerequisite for the sustainable poultry industry. In this regard, genome-editing technologies have been used to control avian diseases, such as avian influenza, Marek’s disease, and avian leukosis [1][2][3]. In some cases, the effective prevention of avian viral diseases has been achieved by the regulation of virus-specific receptors. For example, a subgroup of ALV can lead to cancer in chickens, and this could be prevented by the regulation of host specific ALV receptors [4]. The precise genome editing of chicken NHE1, TVA, TVB, and TVC (specific receptors of ALV subgroups ALV-J, ALV-A, ALV-B, and ALV-C) has been achieved in the chicken DF1 cell line [5][6][7][8]. Such gene editing effectively reduces viral infection, leading to the development of genome-edited chickens. Beyond the cell level, tryptophan 38 (W38) of NHE1, a critical residue for ALV-J entry, was precisely deleted in a commercial chicken line to produce ALV-J-resistant chicken [9].
In relation to avian influenza virus (AIV), resulting in high mortality rates in birds, the host factor ANP32A, which supports the vPol activity of influenza A virus (IAV) in a species-specific manner, is critical [10][11]. The 99 nucleotides of chicken ANP32A encoding the additional 33 amino acids in birds have been deleted in chicken DF1 cells and viral polymerase (vPol) activity was significantly reduced [2][10][11]. Recently, the critical residues (aspartate 149 and 152) for interactions with viral protein in the additional 33 amino acids of chicken ANP32A were revealed [12]. In addition, chickens lost Retinoic inducible gene 1 (RIG-I), a major IAV RNA sensor in mammals; they only harbor Melanoma differentiation associated protein 5 (MDA5), a member of RIG-I like receptor (RLR) family [13]. To gain resistance against IAV, duck RIG-I was introduced into the chicken DF-1 cells [13][14]. Furthermore, the chicken MDA5 C terminal domain (CTD) was replaced with that of RIG-I, resulting in greater inhibition of viral proliferation than that of wild-type chicken MDA5 [15]. Genome editing at these loci could be an effective strategy for the control of avian influenza in a host-specific manner.
Another model of avian disease control has also been proposed for Marek’s disease viruses (MDVs), a lymphotropic α-herpesvirus associated with T-cell lymphoma that induces asymmetric paralysis of the limbs, depression, and death. The targeted disruption of genes essential for MDV replication suggests that effective disease control is possible [3]. Recently, transgenic chickens expressing both Cas9 and gRNA specific to the immediate early infected-cell polypeptide-4 (ICP4) of MDV were produced. The chicken embryonic fibroblasts from transgenic chickens inhibited MDV infection with no effect on herpesvirus of turkeys (HVT) infection [16]. Based on these results, an in-depth understanding of the infection route, replication pathways, and host–virus interactions will provide a basis for the development of an effective genome editing-based disease control model in avian species.

2. Practical Bioreactor System for Recombinant Protein Production in Avian Systems

Transgenic avian bioreactors have great potential for recombinant protein production, including proteins with pharmaceutical and industrial applications in eggs [17][18][19]. Several transgenic avian bioreactors have been reported [20][21][22][23]. However, the conventional system is limited with respect to the quantity of recombinant protein produced. Still, the CRISPR/Cas9 system has made it possible to develop an effective chicken bioreactor system for large-scale production in eggs. Oishi et al. inserted human interferon-β (IFN-β) into the chicken ovalbumin gene via the CRISPR/Cas9 system to produce high levels of recombinant protein (3.5 mg/mL) in chicken egg whites [24]. Human interferon α 2a and porcine colony stimulating factor 1 (CSF1) fused with the Fc region was produced in transgenic chicken eggs and recombinant proteins produced from the chicken bioreactor were easily purified and showed comparable biological functions to those of recombinant proteins produced by other systems [25]. Kim et al. produced anti-cancer monoclonal antibodies against the CD20 with greater Fc effector function in chicken egg whites compared to a commercial counterpart [20]. In addition, gene encoding adiponectin (ADPN), a hormone derived from adipose tissue that can be used to treat insulin resistance, was precisely integrated into the Ovalbumin (OVA) by CRISPR/Cas9 system. The gene-edited chicken expressed high amount of high-molecular-weight (HMW) ADPN, considered to be a more active form [26]. Recently, the GFP gene was inserted into chicken ovalbumin (OVA) gene and system for evaluating protein production in a chicken bioreactor using young chicks was established. This system measured GFP expression in the oviduct of 3-week-old chicks after treatment with an estrogen agonist, diethylstilbestrol (DES) [27]. These results provide a basis for the development of an ideal animal bioreactor that overcomes issues related to yield.
Recombinant proteins from the chicken oviduct derived from egg bioreactor systems with unique post-translational modifications related to N-glycan species terminated with high mannose with a core afucosylated form have been reported [21]. Based on these characteristics, an enzyme produced by transgenic chickens was developed for enzyme replacement therapy for Gaucher disease [28]. As enzymes for the treatment of lysosomal storage diseases, including Gaucher disease, Pompe disease, and Fabry disease, are taken up by mannose receptors, the terminal mannosylation of these recombinant enzymes is critical for efficacy [29][30][31][32]. In this respect, recombinant proteins derived from chicken fallopian tubes are suitable bioreactors to produce these enzymes. Alternatively, the afucosylation of recombinant proteins produced in transgenic chickens can be an effective system for the production of anti-cancer antibodies. N-glycan afucosylation of the Fc domain affects the antibody-dependent cellular cytotoxicity of therapeutic antibodies [33]. Accordingly, levels of antibody-dependent cellular cytotoxicity of antibodies derived from transgenic chickens are significantly higher than those of control chickens [20][21]. Overall, an effective genome-edited avian bioreactor model obtained by CRISPR/Cas9-mediated target gene insertion could be an innovative approach to recombinant protein production for various purposes.

3. Genome-Edited Birds as Scientific Models

The modification of the avian genome provided a basis for the identification of specific gene functions and the development of genome-edited birds as scientific models. For example, recombination activating gene 1 (RAG1) gene was precisely disrupted by CRISPR/Cas9 to obtain chickens lacking mature B and T cells. These chickens could be used to study various lymphocytes in the absence of B and T cells and to study a wide range of diseases, such as cancer and viral infection [34]. In particular, chickens can spontaneously develop ovarian cancer [35]. Thus, RAG1-deficient chickens could be an effective model for studying ovarian cancer. In addition, the GFP gene was precisely inserted into chicken DAZL, a germ cell-specific marker in chicken, to trace germ cells from E2.5 to 1-week post-hatching. Using this model, sex-specific developmental stages and trajectories of chicken germ cells were identified and evolutionary conserved or species-specific genes involved in germ cell development were analyzed [36]. Furthermore, the PR domain zinc finger protein 14 (PRDM14) gene, a critical factor for PGC development in mice, was disrupted in PGCs by inserting eGFP gene via the CRISPR/Cas9 system to evaluate the important roles of PRDM14 in early chicken development [37]. Similarly, double sex and mab-3-related transcription factor 1 (DMRT1) was precisely deleted in chicken, revealing that this gene is one of the most important factors for testis development, while other factors, including sex hormones and DMRT1 gene networks, are key factors for sex determination [38][39].
The zebra finch is a promising bird for neurobiological studies. The Forkhead box protein 2 (FOXP2) mutations in humans lead to developmental verbal dyspraxia (DVD) and lentivirus mediated FOXP2 gene knockdown in zebra finch results in abnormal speech production [40][41]. Transgenic zebra finches carrying the GFP gene under the control of the human ubiquitin-C promoter were generated and GFP-expressing cells located in the forebrain could be traced and analyzed [42][43]. Gonadal PGCs of zebra finch are heterogenous, and the signaling pathways contributing to their development differ from those of chickens [44][45]. In addition, a retrovirus-mediated immortalized zebra finch fibroblast cell line was established and the Sox9 gene was knocked out using CRISPR/Cas9 [46]. Collectively, the generation of transgenic zebra finch models would be facilitated by PGC studies, and the utilization of immortalized cell line; such models are expected to contribute substantially to the understanding the mechanism underlying vocal learning in the near future.

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Subjects: Agriculture, Dairy & Animal Science
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Young-Min Kim
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Seung-Je Woo
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Jae Yong Han
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