Evaluation of CRISPR-Based Approaches for Precision Poultry Gene Modulation and Delivery
Open Access
- Author:
- Chapman, Brittany
- Graduate Program:
- Animal Science (MS)
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- March 13, 2024
- Committee Members:
- Tae Hyun Kim, Thesis Advisor/Co-Advisor
Jason Laurence Rasgon, Committee Member
Adele Turzillo, Program Head/Chair
Francisco Javier Diaz, Committee Member - Keywords:
- CRISPR
Gene Editing
ReMOT
CRISPRa/i
dCas9
QVtg - Abstract:
- The integration of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the CRISPR-associated protein 9 (Cas9) system has expanded the applications of CRISPR beyond DNA cleavage, enabling versatile genetic manipulations. In order to fully harness the potential of CRISPR/Cas9 technology within avian systems, a comprehensive assessment of both modulation strategies and delivery methods is imperative. Evaluating CRISPR-based modulation methods in avian genomes presents a promising approach for achieving targeted genetic modulations. Furthermore, the optimization of delivery methods is paramount for enhancing the efficiency and applicability of CRISPR in avian species. This thesis investigates both CRISPR-mediated transcriptional regulation and the challenges associated with precise gene delivery in avian species. The effectiveness of CRISPRa (activation) and CRISPRi (repression) was demonstrated in chicken DF-1 cells using different effector domains. Four genes were targeted, based on gene expression relevant to DF-1 cells and FPKM values. Multiple gRNAs were developed for each gene, targeting various regulatory elements within the genome. Significant gene upregulation and downregulation were observed, highlighting the adaptability of CRISPRa and CRISPRi toolkits for targeted transcriptional modulation in the chicken genome. Additionally, this thesis explores the applicability of Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) technology for delivery of CRISPR/Cas system in avian species. Initially, visual detection of green fluorescence in the ovary was attempted after injecting adult female quail with QVtg-EGFP protein. Visualization was unsuccessful, and a western blot was employed to attempt to detect protein in samples from ovary and liver samples collected from the injected quail. However, no protein was detected in the samples. The findings emphasize the importance of optimizing the ReMOT Control delivery system for successful gene editing in quail as a research model, providing valuable insights into the complexities of gene delivery in avian species and underscoring the need for continued research to unlock the transformative potential of precise and efficient gene editing in the poultry industry. The assessment of CRISPRa/i technologies and ReMOT Control technologies in avian studies not only serves as a current research tool, but also holds potential for advancing the understanding of avian biology and enhancing the poultry industry, pending further optimization of these tools.