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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