Progress 04/01/24 to 03/31/25
Outputs
Target Audience: Students and researchers in animal and poultry breeding, genetics, and genomics, including undergraduate, graduate, and postdoctoral scholars. Academics and educators in animal science, veterinary science, and agricultural studies. Commercial poultry and livestock breeders focused on improving productivity, disease resistance, and sustainability. Biotechnology and agribusiness professionals working with genetic improvement, artificial insemination, and embryo transfer technologies. Regulatory agencies and policymakers in animal agriculture, genetic research, and food security. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?1. Attendance at PAG32 and Associated Workshops Students attended PAG32 (Plant & Animal Genome Conference 32), a major event in genomics that focused on the latest research and technological advancements in both plant and animal genomics. Within PAG32, students participated in several specialized workshops, which provided in-depth training on cutting-edge topics that aligned with the goals of the project. Key workshops included: Poultry Workshop: This workshop focused on advancements in chicken genomics and gene editing, providing insights into poultry-specific research, functional genomics, and the application of CRISPR technologies in agricultural settings. FAANG Workshop: The FAANG (Functional Annotation of Animal Genomes) workshop concentrated on the functional annotation of animal genomes, with a special emphasis on domesticated animals like chickens. Students learned new methodologies for annotating genomes and explored the role of non-coding regions in gene regulation, which directly aligns with the project's objectives of CRISPR-based gene activation and repression. FarmGTEx Workshop: The FarmGTEx (Functional Annotation of Livestock Genomes) workshop provided training on identifying genetic traits in livestock. Students learned how genomic data can be applied to enhance traits such as growth and disease resistance, complementing the functional genomics approaches being explored in the project. These workshops not only enhanced the students' knowledge but also provided valuable networking and collaboration opportunities. Students interacted with leading researchers and experts in the fields of poultry genomics, functional genomics, and animal breeding, which fostered future collaborations and exposure to a wide range of scientific research in animal genomics. 2. Attendance at the Genome Engineering: CRISPR Frontiers 2024 Conference In addition to PAG32, students had the opportunity to attend the Genome Engineering: CRISPR Frontiers 2024 conference, a key event in the CRISPR research community. This conference brought together influential leaders, including Jennifer Doudnaand others, who are pioneers in the development of CRISPR technologies. Exposure to Leading Experts: Students attended keynote presentations and panel discussions, gaining insights into the latest advances in CRISPR technology, gene editing, and molecular genetics. These sessions helped them stay at the forefront of CRISPR-related research. Networking Opportunities: The conference also offered numerous opportunities for students to connect with renowned scientists, researchers, and innovators, which were invaluable for future career development and potential collaborations in genomics and biotechnology. Cutting-edge Topics: Students participated in sessions covering a broad range of CRISPR-related topics, including next-generation CRISPR tools, applications in medicine and agriculture, ethical considerations, and the future of gene editing. This exposure provided students with a comprehensive understanding of the CRISPR landscape and its implications for research and applications in various fields. 3. On-Campus Professional Development Workshops Beyond the conferences, students participated in on-campus professional development workshops that complemented their research training. These workshops were designed to build essential skills for their academic and professional growth: Scientific Writing and Publishing: Students learned how to effectively write research papers, grants, and proposals, with a focus on how to communicate complex scientific findings to a broad audience. Career Development: Workshops provided guidance on navigating career paths in academia, research, industry, and biotechnology. Students were equipped with skills for job applications, interviews, and career planning. Data Analysis and Computational Tools: Students gained hands-on experience with data analysis tools and software commonly used in genomic research, enhancing their ability to work with large, complex biological datasets and apply bioinformatics techniques. How have the results been disseminated to communities of interest? Scientific Talks at Conferences: Presentations at major conferences like PAG32, engaging a global audience of researchers and industry professionals. Scientific Networks: Sharing results through professional societies (e.g., ISAG, PSA) and online networks, fostering collaboration and knowledge exchange. Peer-Reviewed Publications: Publishing in relevant journalsto ensure validated, widespread access to research findings. What do you plan to do during the next reporting period to accomplish the goals? Aim 3: Focus on establishing and optimizing CRISPRa and CRISPRi high-throughput single-cell based CRISPR functional screening (scCRISPR-seq) for precise gene regulation analysis at the single-cell level. Generate More Data: Conduct experiments to generate data on gene activation and repression efficiency, refining protocols for improved reproducibility and scalability. Refine Protocols: Continuously fine-tune CRISPRa and CRISPRi systems for optimal performance in high-throughput, single-cell applications. Hold Workshop: Organize a workshop to present progress, share methodologies, and receive feedback from experts in CRISPR technology and functional genomics. Attend ISAG2025, TARC2025 & PAG2026: Present findings and engage with the global scientific community at ISAG2025 (International Society for Animal Genetics), TARC2025 (Transgenic Animal Research Conference), and PAG2026 (Plant & Animal Genomes Conference), focusing on CRISPR-based gene regulation techniques and advances in transgenic research.
Impacts
What was accomplished under these goals?
Aim 1: Establish and Optimize In Vitro CRISPR-based Transcription Activation (CRISPRa) Systems in Different Lineages of Chicken Cell Lines Under Aim 1, CRISPRa systems were successfully developed and optimized for transcriptional activation in various chicken cell lines. These systems used catalytically dead Cas9 (dCas9) fused with transcriptional activators, including dCas9-VPR, dCas9-p300, and dCas9-KRAB. The CRISPRa systems were stably integrated into the following chicken cell lines: DF1 (Immortalized embryonic fibroblasts): The systems were optimized for efficient gene activation in DF1 cells, demonstrating practical use for gene functional studies. DT40 (B-cell lymphoma): CRISPRa systems were tested to activate genes related to immune cell functions, with successful results. LMH (Liver epithelial cells): The CRISPRa constructs were applied to activate tissue-specific genes in LMH cells, showing the potential for liver-related research. Through this work, optimized protocols for CRISPRa in these cell lines were established, and resources (plasmids and protocols) are being prepared for broader distribution within the research community. Aim 2: Establish and Optimize In Vitro CRISPR-based Transcription Repression (CRISPRi) Systems in Different Lineages of Chicken Cell Lines For Aim 2, the project successfully established CRISPRi systems to repress gene expression. Using dCas9 fused with KRAB, these systems were integrated into the same chicken cell lines from Aim 1: DF1 Cell Line: CRISPRi systems were optimized to efficiently silence genes, validated by reporter assays that confirmed target repression. DT40 and LMH Cell Lines: CRISPRi was applied to these cell lines, effectively repressing genes of interest in both immune and liver cells, allowing further study of gene function in specific biological contexts. The progress made under Aim 2 has led to the creation of reliable CRISPRi tools, with established protocols for gene silencing in these chicken cell lines. These tools are now available for use in gene function studies and other applications requiring controlled gene repression. ?Aim 3 is planned for Year 3.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2025
Citation:
Lee, H. J., Han, J. H., Chapman, B., Jung, K. M., Rudd, I., Han, J. Y., & Kim, T. H. (2025). A comprehensive transcriptional profiling of developing gonads reveals the role of TGF? signaling in female gonadal asymmetry in chickens. Poultry Science, 104(4), 104932
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2025
Citation:
Kim, T. H. (2025). Development of CRISPR-based in vitro resources for studying the chicken genome. Poultry Workshop, Plant & Animal Genome Conference 32 (PAG32).
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2025
Citation:
Kim, T. H. (2025). From FAANG data to function: CRISPR tools for understanding chicken non-coding regulatory elements. FAANG Workshop, Plant & Animal Genome Conference 32 (PAG32).
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2025
Citation:
Han, J. H., Lee, H., & Kim, T. H. (2025). CRISPR-mediated activation for characterizing enhancer elements in the chicken genome (No. P260). Proceedings of the Plant & Animal Genome Conference 32 (PAG32).
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2025
Citation:
Weaver, E. A., Schmidt, G. E., Han, J. H., & Kim, T. H. (2025). Targeted gene silencing and activation in avian cells: Insights into immune signaling using a dual CRISPR a/I system (No. P261). Proceedings of the Plant & Animal Genome Conference 32 (PAG32).
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2025
Citation:
Schmidt, G. E., Weaver, E. A., Han, J. H., & Kim, T. H. (2025). Function of IRF9 in the chicken anti-viral immune response (No. P278). Proceedings of the Plant & Animal Genome Conference 32 (PAG32).
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Kim, T. H. (2024). CRISPR-powered exploration of the chicken genome. CCAT 2024.
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Progress 04/01/23 to 03/31/24
Outputs
Target Audience:- Students and researchers in animal breeding, genetics and genomics - Commercial livestock breeders Changes/Problems:Despite our concerted efforts, we were unable to successfully engineer CRISPRa or CRISPRi cells using HD11 macrophages. This limitation can be attributed to the inherent challenges associated with manipulating and differentiating macrophage cell lines. Macrophages are known for their innate immune function, which often involves recognizing and responding to foreign nucleic acids like those used in CRISPR-based gene editing. This immune response can lead to the degradation or silencing of CRISPR components, hindering their efficacy. Additionally, macrophages are highly specialized cells with specific differentiation pathways, making it difficult to maintain their desired phenotype while introducing genetic modifications. These factors collectively contribute to the challenges encountered in engineering CRISPR-based cell lines using HD11 macrophages. What opportunities for training and professional development has the project provided?One of our graduate students attended the Transgenic Animal Research Conference XIV, where their exceptional research was recognized with a Graduate Student Fellowship. This award provided them with the unique opportunity to network with leading experts in the field, learn about cutting-edge research, present their own work to a wider audience, and develop their scientific communication skills. By connecting with renowned researchers, staying updated on the latest advancements, showcasing their findings, and honing their communication abilities, our student was able to significantly enhance their professional growth and expand their research horizons. How have the results been disseminated to communities of interest?Our research findings were disseminated through a peer-reviewed publication, contributing to the advancement of the field. Additionally, we presented our results at the prestigious Transgenic Animal Research Conference XIV, where we engaged with leading experts and shared our discoveries with a wider scientific audience. What do you plan to do during the next reporting period to accomplish the goals?Aim 1 & 2 To comprehensively validate our established cell lines, we will conduct rigorous assessments of their genotype, gene expression, and functionality. This will involve employing advanced techniques to ensure the accuracy and reliability of our cell models. Furthermore, we will leverage the FAANG dataset to experimentally identify functional enhancer regions within these cell lines. We plan to present our data at the poultry workshop and FAANG workshop at PAG 2025. ?Aim 3 In parallel, we will initiate the selection of candidate loci for library-based pooled CRISPR screening using scRNAseq. This approach will enable us to systematically interrogate the functional relevance of genes and regulatory elements in a high-throughput manner. By combining CRISPR screening with scRNAseq, we can identify genes and regulatory elements that are specifically involved in distinct cellular states or functions.
Impacts
What was accomplished under these goals?
Aim 1: CRISPRa Cell Line Development Key Achievements: Successful CRISPRa Cell Line Generation: We have successfully established a total of six CRISPRa cell lines through homology-directed repair (HDR)-mediated targeted knock-in of the expression construct. Diverse Cell Types and Effector Combinations: The generated cell lines span a range of cell types, including DF1, DT40, and LMH, and utilize two distinct activator effectors, VPR and p300. Targeted Gene Activation: These CRISPRa cell lines provide a powerful platform for precisely activating genes of interest in various biological contexts. Aim 2: CRISPRi Cell Line Development Key Achievements: Establishment of CRISPRi Cell Lines: We have successfully generated three CRISPRi cell lines using HDR-mediated knock-in of the expression construct. Diverse Cell Types and Effector: These cell lines encompass DF1, DT40, and LMH cell types, and employ the KRAB repressor effector. Targeted Gene Repression: The CRISPRi cell lines offer a valuable tool for precisely silencing genes of interest in various biological systems. Overall, our accomplishments in both Aim 1 and Aim 2 have laid a solid foundation for further development and application of CRISPR-based gene editing technologies.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Han JH, Lee HJ and Kim TH (2023) Characterization of transcriptional enhancers in the chicken genome using CRISPR-mediated activation. Front. Genome Ed. 5:1269115. doi: 10.3389/fgeed.2023.1269115
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Han JH and Kim TH (2023) Identification of potential enhancer regions in chicken genome using CRISPR activation system. Transgenic Animal Research Conference XIV. Tahoe City, California
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