Characterization of transcriptional enhancers in the chicken genome using CRISPR-mediated activation.

DNA regulatory elements intricately control when, where, and how genes are activated. Therefore, understanding the function of these elements could unveil the complexity of the genetic regulation network. Genome-wide significant variants are predominantly found in non-coding regions of DNA, so comprehending the predicted functional regulatory elements is crucial for understanding the biological context of these genomic markers, which can be incorporated into breeding programs. The emergence of CRISPR technology has provided a powerful tool for studying non-coding regulatory elements in genomes. In this study, we leveraged epigenetic data from the Functional Annotation of Animal Genomes project to identify promoter and putative enhancer regions associated with three genes (HBBA, IRF7, and PPARG) in the chicken genome. To identify the enhancer regions, we designed guide RNAs targeting the promoter and candidate enhancer regions and utilized CRISPR activation (CRISPRa) with dCas9-p300 and dCas9-VPR as transcriptional activators in chicken DF-1 cells. By comparing the expression levels of target genes between the promoter activation and the co-activation of the promoter and putative enhancers, we were able to identify functional enhancers that exhibited augmented upregulation. In conclusion, our findings demonstrate the remarkable efficiency of CRISPRa in precisely manipulating the expression of endogenous genes by targeting regulatory elements in the chicken genome, highlighting its potential for functional validation of non-coding regions.

Targeted Modulation of Chicken Genes In Vitro Using CRISPRa and CRISPRi Toolkit.

Engineering of clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated protein 9 (Cas9) system has enabled versatile applications of CRISPR beyond targeted DNA cleavage. Combination of nuclease-deactivated Cas9 (dCas9) and transcriptional effector domains allows activation (CRISPRa) or repression (CRISPRi) of target loci. To demonstrate the effectiveness of the CRISPR-mediated transcriptional regulation in chickens, three CRISPRa (VP64, VPR, and p300) and three CRISPRi (dCas9, dCas9-KRAB, and dCas9-KRAB-MeCP2) systems were tested in chicken DF-1 cells. By introducing guide RNAs (gRNAs) targeting near the transcription start site (TSS) of each gene in CRISPRa and CRISPRi effector domain-expressing chicken DF-1 cell lines, significant gene upregulation was induced in dCas9-VPR and dCas9-VP64 cells, while significant downregulation was observed with dCas9 and dCas9-KRAB. We further investigated the effect of gRNA positions across TSS and discovered that the location of gRNA is an important factor for targeted gene regulation. RNA sequencing analysis of IRF7 CRISPRa and CRISPRi- DF-1 cells revealed the specificity of CRISPRa and CRISPRi-based targeted transcriptional regulation with minimal off-target effects. These findings suggest that the CRISPRa and CRISPRi toolkits are an effective and adaptable platform for studying the chicken genome by targeted transcriptional modulation.

Genome Editing Mediated by Primordial Germ Cell in Chicken.

Genome editing technology has facilitated the studies on exploring specific gene functions in diverse living organisms. The technology has also contributed to creating high-value livestock in industry fields in terms of enhancing productivity or acquiring disease resistance. Particularly, applying genome editing technologies in avian species has been emphasized in both academic and industrial fields due to their unique developmental patterns as well as application possibilities. To accomplish genome editing in avian species, gene integration into chicken primordial germ cell (PGC) genome using a virus or transposition systems has been widely used, and recently developed programmable genome editing technologies including clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas9) systems enable to edit the genetic information precisely for maximizing the application potentials of avian species. In these regards, this chapter will cover the methods for producing genome-edited chickens, particularly by CRISPR/Cas9 technologies allowing targeted gene insertion, gene knockout, and gene tagging.

Dissecting chicken germ cell dynamics by combining a germ cell tracing transgenic chicken model with single-cell RNA sequencing.

Avian germ cells can be distinguished by certain characteristics during development. On the basis of these characteristics, germ cells can be used for germline transmission. However, the dynamic transcriptional landscape of avian germ cells during development is unknown. Here, we used a novel germ-cell-tracing method to monitor and isolate chicken germ cells at different stages of development. We targeted the deleted in azoospermia like (DAZL) gene, a germ-cell-specific marker, to integrate a green fluorescent protein (GFP) reporter gene without affecting endogenous DAZL expression. The resulting transgenic chickens (DAZL::GFP) were used to uncover the dynamic transcriptional landscape of avian germ cells. Single-cell RNA sequencing of 4,752 male and 13,028 female DAZL::GFP germ cells isolated from embryonic day E2.5 to 1 week post-hatch identified sex-specific developmental stages (4 stages in male and 5 stages in female) and trajectories (apoptosis and meiosis paths in female) of chicken germ cells. The male and female trajectories were characterized by a gradual acquisition of stage-specific transcription factor activities. We also identified evolutionary conserved and species-specific gene expression programs during both chicken and human germ-cell development. Collectively, these novel analyses provide mechanistic insights into chicken germ-cell development.

DMRT1 gene disruption alone induces incomplete gonad feminization in chicken.

Compared with the well-described XY sex determination system in mammals, the avian ZW sex determination system is poorly understood. Knockdown and overexpression studies identified doublesex and mab-3-related transcription factor 1 (DMRT1) as the testis-determining gene in chicken. However, the detailed effects of DMRT1 gene disruption from embryonic to adult development are not clear. Herein, we have generated DMRT1-disrupted chickens using the clustered regularly interspaced short palindromic repeats-associated protein 9 system, followed by an analysis of physiological, hormonal, and molecular changes in the genome-modified chickens. In the early stages of male chicken development, disruption of DMRT1 induced gonad feminization with extensive physiological and molecular changes; however, functional feminine reproductivity could not be implemented with disturbed hormone synthesis. Subsequent RNA-sequencing analysis of the DMRT1-disrupted chicken gonads revealed gene networks, including several novel genes linearly and non-linearly associated with DMRT1, which are involved in gonad feminization. By comparing the gonads of wild type with the genome-modified chickens, a set of genes were identified that is involved in the ZW sex determination system independent of DMRT1. Our results extend beyond the Z-dosage hypothesis to provide further information about the avian ZW sex determination system and epigenetic effects of gonad feminization.

Asp149 and Asp152 in chicken and human ANP32A play an essential role in the interaction with influenza viral polymerase.

The acidic nuclear phosphoprotein 32 family member A (ANP32A) is a cellular host factor that determines the host tropism of the viral polymerase (vPol) of avian influenza viruses (AIVs). Compared with human ANP32A (hANP32A), chicken ANP32A contains an additional 33 amino acid residues (176-208) duplicated from amino acid residues 149-175 (27 residues), suggesting that these residues could be involved in increasing vPol activity by strengthening interactions between ANP32A and vPol. However, the molecular interactions and functional roles of the 27 residues within hANP32A during AIV vPol activity remain unclear. Here, we examined the functional role of 27 residues of hANP32A based on comparisons with other human (h) ANP32 family members. It was notable that unlike hANP32A and hANP32B, hANP32C could not support vPol activity or replication of AIVs, despite the fact that hANP32C shares a higher sequence identity with hANP32A than hANP32B. Pairwise comparison between hANP32A and hANP32C revealed that Asp149 (D149) and Asp152 (D152) are involved in hydrogen bonding and electrostatic interactions, respectively, which support vPol activity. Mutation of these residues reduced the interaction between hANP32A and vPol. Finally, we demonstrated that precise substitution of the identified residues within chicken ANP32A via homology-directed repair using the CRISPR/Cas9 system resulted in a marked reduction of viral replication in chicken cells. These results increase our understanding of ANP32A function and may facilitate the development of AIV-resistant chickens via precise modification of residues within ANP32A.

Establishment of a genetically engineered chicken DF-1 cell line for efficient amplification of influenza viruses in the absence of trypsin.

BACKGROUND: The initial step of influenza infection is binding of the virus to specific sialic acid receptors expressed by host cells. This is followed by cell entry via endocytosis. Cleavage of the influenza virus hemagglutinin (HA) protein is critical for infection; this is performed by host cell proteases during viral replication. In cell culture systems, HA is cleaved by trypsin added to the culture medium. The vast majority of established cell lines are mammalian. RESULTS: In the present study, we generated genetically engineered chicken DF-1 cell lines overexpressing transmembrane protease, serine 2 (TMPRSS2, which cleaves HA), ST3 beta-galactoside alpha-2,3-sialyltransferase 1 (ST3GAL1, which plays a role in synthesis of α-2,3 linked sialic acids to which avian-adapted viruses bind preferentially), or both. We found that overexpression of TMPRSS2 supports the virus life cycle by cleaving HA. Furthermore, we found that overexpression of ST3GAL1 increased the viral titer. Finally, we showed that overexpression of both TMPRSS2 and ST3GAL1 increased the final viral titer due to enhanced support of viral replication and prolonged viability of the cells. In addition, overexpression of these genes of interest had no effect on cell proliferation and viability. CONCLUSIONS: Taken together, the results indicate that these engineered cells could be used as a cell-based system to propagate influenza virus efficiently in the absence of trypsin. Further studies on influenza virus interactions with chicken cell host factors could be studied without the effect of trypsin on cells.

Highly elevated base excision repair pathway in primordial germ cells causes low base editing activity in chickens.

Base editing technology enables the generation of precisely genome-modified animal models. In this study, we applied base editing to chicken, an important livestock animal in the fields of agriculture, nutrition, and research through primordial germ cell (PGC)-mediated germline transmission. Using this approach, we successfully produced two genome-modified chicken lines harboring mutations in the genes encoding ovotransferrin (TF) and myostatin (MSTN); however, only 55.5% and 35.7% of genome-modified chickens had the desired base substitutions in TF and MSTN, respectively. To explain the low base-editing activity, we performed molecular analysis to compare DNA repair pathways between PGCs and the chicken fibroblast cell line DF-1. The results revealed that base excision repair (BER)-related genes were significantly elevated in PGCs relative to DF-1 cells. Subsequent functional studies confirmed that the editing activity could be regulated by modulating the expression of uracil N-glycosylase (UNG), an upstream gene of the BER pathway. Collectively, our findings indicate that the distinct DNA repair property of chicken PGCs causes low editing activity during genome modification, however, modulation of BER functions could promote the production of genome-modified organisms with the desired genotypes.

Host-Specific Restriction of Avian Influenza Virus Caused by Differential Dynamics of ANP32 Family Members.

BACKGROUND: Influenza viruses must utilize host factors to complete their lifecycle. Species-specific differences in host factors between birds and mammals mean that avian influenza viruses (AIVs) replicate well in avian hosts but not in human hosts. Acidic nuclear phosphoprotein 32 family member A (ANP32A) has been identified as the host restriction factor for the viral polymerase (vPol) activity of AIVs. The ANP32A belongs to the conserved ANP32 family, the functional roles of which during viral replication remain unclear. METHODS: In this study, we targeted chicken ANP32A using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome editing to examine the functional roles of ANP32A and other members of the ANP32 family. RESULTS: We showed that chicken ANP32A only, not ANP32B and ANP32E, plays a pivotal role in supporting vPol activity of AIVs. Furthermore, we found that the human ANP32C, ANP32D, and ANP32E have suppressive effects on vPol activity in contrast to human ANP32A and ANP32B. CONCLUSIONS: Chicken and human ANP32 family members had different effects on vPol activity, suggesting that species-specific vPol activity of AIVs could be caused by the differential functions and overall competency of ANP32 family members.

Identification and characterization of primordial germ cells in a vocal learning Neoaves species, the zebra finch.

The zebra finch has been used as a valuable vocal learning animal model for human spoken language. It is representative of vocal learning songbirds specifically, which comprise half of all bird species, and of Neoaves broadly, which comprise 95% of all bird species. Although transgenesis in the zebra finch has been accomplished, it is with a very low efficiency of germ-line transmission and far from the efficiency with a more genetically tractable but vocal nonlearning species, the chicken (a Galloanseriformes). To improve germ-line transmission in the zebra finch, we identified and characterized its primordial germ cells (PGCs) and compared them with chicken. We found striking differences between the 2 species, including that zebra finch PGCs were more numerous, more widely distributed in early embryos before colonization into the gonads, had slower timing of colonization, and had a different developmental gene-expression program. We improved conditions for isolating and culturing zebra finch PGCs in vitro and were able to transfect them with gene-expression vectors and incorporate them into the gonads of host embryos. Our findings demonstrate important differences in the PGCs of the zebra finch and advance the first stage of creating PGC-mediated germ-line transgenics of a vocal learning species.-Jung, K. M., Kim, Y. M., Keyte, A. L., Biegler, M. T., Rengaraj, D., Lee, H. J., Mello, C. V., Velho, T. A. F., Fedrigo, O., Haase, B., Jarvis, E. D., Han, J. Y. Identification and characterization of primordial germ cells in a vocal learning Neoaves species, the zebra finch.

Sequential disruption of ALV host receptor genes reveals no sharing of receptors between ALV subgroups A, B, and J.

BACKGROUND: Previously, we showed that targeted disruption of viral receptor genes in avian leukosis virus (ALV) subgroups using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9))-based genome editing confers resistance to ALV subgroups B and J. Here, we used the same strategy to target the receptor expressed by ALV subgroup A (TVA) and generate chicken cells resistant to infection by this virus. RESULTS: CRISPR/Cas9-based disruption of exon 2 within the tva gene of DF-1 fibroblasts conferred resistance to infection by ALV subgroup A regardless of whether frameshift mutations were introduced during editing. Conversely, overexpression of the wild-type TVA receptor (wtTVA) by tva-modified DF-1 clones restored susceptibility to ALV subgroup A. The results confirm that exon 2, which contains the low-density lipoprotein receptor class A domain of TVA, is critical for virus entry. Furthermore, we sequentially modified DF-1 cells by editing the tva, tvb, and Na+/H+ exchange 1 (chNHE1) genes, which are the specific receptors for ALV subgroups A, B, and J, respectively. CONCLUSIONS: Simultaneous editing of multiple receptors to block infection by different subgroups of ALV confirmed that ALV subgroups A, B, and J do not share host receptors. This strategy could be used to generate cells resistant to multiple viral pathogens that use distinct receptors for cell entry.

Targeted gene insertion into Z chromosome of chicken primordial germ cells for avian sexing model development.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) have facilitated the production of genome-edited animals for use as models. Because of their unique developmental system, avian species offer many advantages as model vertebrates. Here, we report the development of novel chicken models using the CRISPR/Cas9-mediated nonhomologous end joining repair pathway in chicken primordial germ cells (PGCs). Through the introduction of a donor plasmid containing short guide RNA recognition sequences and CRISPR/Cas9 plasmids into chicken PGCs, exogenous genes of donor plasmids were precisely inserted into target loci, and production of transgenic chickens was accomplished through subsequent transplantation of the Z chromosome-targeted PGCs. Using this method, we successfully accomplished the targeted gene insertion to the chicken sex Z chromosome without detected off-target effects. The genome-modified chickens robustly expressed green fluorescent protein from the Z chromosome, which could then be used for easy sex identification during embryogenesis. Our results suggest that this powerful genome-editing method could be used to develop many chicken models and should significantly expand the application of genome-modified avians.-Lee, H. J., Yoon, J. W., Jung, K. M., Kim, Y. M., Park, J. S., Lee, K. Y., Park, K. J., Hwang, Y. S., Park, Y. H., Rengaraj, D., Han, J. Y. Targeted gene insertion into Z chromosome of chicken primordial germ cells for avian sexing model development.

The transgenic chicken derived anti-CD20 monoclonal antibodies exhibits greater anti-cancer therapeutic potential with enhanced Fc effector functions.

Modern genetic techniques, enable the use of animal bioreactor systems for the production and functional enhancement of anti-cancer antibodies. Chicken is the most efficient animal bioreactor for the production of anti-cancer antibodies because of its relatively short generation time, plentiful reproductive capacity, and daily deposition in the egg white. Although several studies have focused on the production of anti-cancer antibodies in egg white, in-depth studies of the biological activity and physiological characteristics of transgenic chicken-derived anti-cancer antibodies have not been fully carried out. Here, we report the production of an anti-cancer monoclonal antibody against the CD20 protein from egg whites of transgenic hens, and validated the bio-functional activity of the protein in B-lymphoma and B-lymphoblast cells. Quantitative analysis showed that deposition of the chickenised CD20 monoclonal antibody (cCD20 mAb) from transgenic chickens increased in successive generations and with increasing transgene copy number. Ultra-performance liquid chromatography (UPLC) tandem mass spectrometry (LC/MS/MS) analysis showed that the cCD20 mAb exhibited 14 N-glycan patterns with high-mannose, afucosylation and terminal galactosylation. The cCD20 mAb did not exhibit significantly improved Fab-binding affinity, but showed markedly enhanced Fc-related functions, including complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) compared to commercial rituximab, a chimeric mAb against CD20. Our results suggest that the transgenic chicken bioreactor is an efficient system for producing anti-cancer therapeutic antibodies with enhanced Fc effector functions.

The transcriptome of early chicken embryos reveals signaling pathways governing rapid asymmetric cellularization and lineage segregation.

The phylogenomics and comparative functional genomics of avian species were investigated in the Bird 10,000 Genomes (B10K) project because of the important evolutionary position of birds and their value as a research model. However, the systematic profiling of transcriptional changes prior to oviposition has not been investigated in avian species because of the practical difficulties in obtaining pre-oviposited eggs. In this study, a total of 137 pre-oviposited embryos were collected from hen ovaries and oviducts and subjected to RNA-sequencing analyses. Two waves of chicken zygotic genome activation (ZGA) were observed. Functionally distinct developmental programs involving Notch, MAPK, Wnt and TGFß signaling were separately detected during cleavage and area pellucida formation. Furthermore, the early stages of chicken development were compared with the human and mouse counterparts, highlighting chicken-specific signaling pathways and gradually analogous gene expression via ZGA. These findings provide a genome-wide understanding of avian embryogenesis and comparisons among amniotes.

Chicken NANOG self-associates via a novel folding-upon-binding mechanism.

NANOG plays a pivotal role in pluripotency acquisition and lineage specification in higher vertebrates, and its expression is restricted to primordial germ cells (PGCs) during early embryonic development. Mammalian NANOG self-associates via conserved tryptophan-repeat motifs in the C-terminal domain (CTD) to maintain pluripotency. Avian NANOG, however, lacks the conserved motifs, and the molecular mechanism underlying the biologic function is not clearly understood. Here, using spectroscopic and biochemical methods as well as cell-based assays, we report that chicken NANOG (cNANOG) oligomerizes through its CTD via a novel folding-upon-binding mechanism. The CTD of cNANOG is disordered as a monomer and associates into an α-helical multimer driven by intermolecular hydrophobic interactions. Mutation of key aromatic residues in the CTD abrogates the self-association, leading to a loss of the proliferation of chicken PGCs and blastoderm cells. Our results demonstrate that the CTD of cNANOG belongs to a novel IDP that switches into a helical oligomer via self-association, enabling the maintenance of PGCs and blastoderm cells.-Choi, H. J., Kim, I., Lee, H. J., Park, Y. H., Suh, J.-Y., Han, J. Y. Chicken NANOG self-associates via a novel folding-upon-binding mechanism.

The dynamic development of germ cells during chicken embryogenesis.

Appropriate regulation of cell proliferation during embryogenesis is crucial for the maintenance of germness. An in-depth understanding of germ cell developmental processes may yield valuable information on germ cell biology and applied sciences. However, direct evidences about germ cell proliferation and cell cycling during avian embryonic development has not been well-studied. Thus, we explored chicken germ cell dynamics during embryonic development via flow cytometry employing a germ cell-specific anti-cVASA antibody (the chicken VASA homolog is termed CVH) and propidium iodide staining. The numbers of male germ cells increased significantly during early embryonic development, but proliferation was decreased significantly with accumulation at the G0/G1 phase after embryonic d 14 (E.14), indicating initiation of mitotic arrest in the testis. On the other hand, the number of female germ cells increased significantly throughout embryogenesis, and proliferating cells were continuously evident in the ovary to the time of hatching, although gradual accumulation of cells at the G2/M phase was also evident. 5-ethynyl-2΄-deoxyuridine (EdU) incorporation analysis revealed that populations of mitotically active germ cells existed in both sexes during late embryogenesis, indicating either the maintenance of stem cell populations, or asynchronous meiosis. Collectively, these results indicate that chicken germ cells exhibited conserved developmental processes that were clearly sexually dimorphic.

Genome Modification Technologies and Their Applications in Avian Species.

The rapid development of genome modification technology has provided many great benefits in diverse areas of research and industry. Genome modification technologies have also been actively used in a variety of research areas and fields of industry in avian species. Transgenic technologies such as lentiviral systems and piggyBac transposition have been used to produce transgenic birds for diverse purposes. In recent years, newly developed programmable genome editing tools such as transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) have also been successfully adopted in avian systems with primordial germ cell (PGC)-mediated genome modification. These genome modification technologies are expected to be applied to practical uses beyond system development itself. The technologies could be used to enhance economic traits in poultry such as acquiring a disease resistance or producing functional proteins in eggs. Furthermore, novel avian models of human diseases or embryonic development could also be established for research purposes. In this review, we discuss diverse genome modification technologies used in avian species, and future applications of avian biotechnology.

Acquisition of resistance to avian leukosis virus subgroup B through mutations on tvb cysteine-rich domains in DF-1 chicken fibroblasts.

Avian leukosis virus (ALV) is a retrovirus that causes tumors in avian species, and its vertical and horizontal transmission in poultry flocks results in enormous economic losses. Despite the discovery of specific host receptors, there have been few reports on the modulation of viral susceptibility via genetic modification. We therefore engineered acquired resistance to ALV subgroup B using CRISPR/Cas9-mediated genome editing technology in DF-1 chicken fibroblasts. Using this method, we efficiently modified the tumor virus locus B (tvb) gene, encoding the TVB receptor, which is essential for ALV subgroup B entry into host cells. By expanding individual DF-1 clones, we established that artificially generated premature stop codons in the cysteine-rich domain (CRD) of TVB receptor confer resistance to ALV subgroup B. Furthermore, we found that a cysteine residue (C80) of CRD2 plays a crucial role in ALV subgroup B entry. These results suggest that CRISPR/Cas9-mediated genome editing can be used to efficiently modify avian cells and establish novel chicken cell lines with resistance to viral infection.

Precise gene editing of chicken Na+/H+ exchange type 1 (chNHE1) confers resistance to avian leukosis virus subgroup J (ALV-J).

Avian leukosis virus subgroup J (ALV-J), first isolated in the late 1980s, has caused economic losses to the poultry industry in many countries. As all chicken lines studied to date are susceptible to ALV infection, there is enormous interest in developing resistant chicken lines. The ALV-J receptor, chicken Na+/H+ exchange 1 (chNHE1) and the critical amino acid sequences involved in viral attachment and entry have already been characterized. However, there are no reported attempts to induce resistance to the virus by targeted genome modification of the receptor sequences. In an attempt to induce resistance to ALV-J infection, we used clustered regularly interspaced short palindromic repeats (CRISPR)-associated (CRISPR/Cas9)-based genome editing approaches to modify critical residues of the chNHE1 receptor in chicken cells. The susceptibility of the modified cell lines to ALV-J infection was examined using enhanced green fluorescent protein (EGFP)-expressing marker viruses. We showed that modifying the chNHE1 receptor by artificially generating a premature stop codon induced absolute resistance to viral infection, with mutations of the tryptophan residue at position 38 (Trp38) being very critical. Single-stranded oligodeoxynucleotide (ssODN)-mediated targeted recombination of the Trp38 region revealed that deletions involving the Trp38 residue were most effective in conferring resistance to ALV-J. Moreover, protein structure analysis of the chNHE1 receptor sequence suggested that its intrinsically disordered region undergoes local conformational changes through genetic alteration. Collectively, these results demonstrate that targeted mutations on chNHE1 alter the susceptibility to ALV-J and the technique is expected to contribute to develop disease-resistant chicken lines.

Genome Editing Mediated by Primordial Germ Cell in Chicken.

Rapid development of genome editing technology has facilitated the studies on exploring specific gene functions and establishment of model animals. In livestock, the technology has contributed to create high value in industry fields, e.g., enhancing productivity or acquiring the resistance against disease. Meanwhile, genome editing in avian species has been emphasized because of their applicable possibilities in terms of highly productive chickens, disease-controlled avian lines, and development of novel biological models. Induction of exogenous gene using virus system or transposition in chicken primordial germ cells (PGCs) has been widely used for producing transgenic chicken, and recently developed programmable genome editing (PGE) technologies such as tale transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas9) are expected to maximize the applicable potentials of avian species. In this regard, this chapter will cover the methods for producing genome-edited chicken by piggyBac transposition and gene targeting technology, TALEN, and CRISPR/Cas9.