Evaluation of CRISPR Activation Toolkit for Targeted Activation of Regulatory Elements in Chickens
Open Access
- Author:
- Han, Jeong Hoon
- Graduate Program:
- Animal Science
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- May 22, 2023
- Committee Members:
- Adele Turzillo, Program Head/Chair
Tae Hyun Kim, Thesis Advisor/Co-Advisor
Wansheng Liu, Committee Member
Ramesh Ramachandran, Committee Member - Keywords:
- Chicken
CRISPR
dCas9
Activation - Abstract:
- Regulatory elements are critical in controlling when, where, and to what extent a gene is expressed. The emergence of CRISPR/Cas9 technology has revolutionized genetic research by facilitating precise manipulation of these elements. Among its applications, CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) enable targeted upregulation or downregulation of gene expression by modifying regulatory elements. Although CRISPRa and CRISPRi system has succeeded in other model systems for gene activation, its potential in avian species remains largely unexplored. This study utilized chicken DF-1 cells to investigate the applicability of CRISPR-mediated transcriptional regulation. Three different CRISPRa systems (dCas9-VP64, dCas9-VPR, and dCas9-p300) and three different CRISPRi systems (dCas9, dCas9-KRAB, and dCas9-KRAB-MeCP2) were tested. Guide RNAs (gRNAs) were designed to target regions near the transcription start site (TSS) of specific genes in chicken DF-1 cell lines expressing the CRISPRa and CRISPRi effector domains. Four different genes (HMGA1, SMARCB1, IRF7, and PPARG) were selected based on their FPKM values, and each cell line was tested with different genes. The promoter region was found to be most effective in the dCas9-VPR and dCas9-VP64 cell lines, while significant downregulation was observed with the dCas9 and dCas9-KRAB cell lines. This study also investigated the influence of gRNA positions relative to the TSS revealing the crucial role of gRNA location in targeted gene regulation. RNA sequencing analysis of IRF7 CRISPRa and CRISPRi DF-1 cells exhibited specific transcriptional regulation with minimal off-target effects. Furthermore, the study aimed to identify potential enhancer regions in the HBBA, IRF7, and PPARG genes. In order to accomplish this, three to four guide RNAs (gRNAs) were designed in proximity to the novel enhancer regions identified using epigenetic data from the Functional Annotation of ANimal Genomes (FAANG) database. This research highlights the potential of CRISPRa-based gene activation in identifying and characterizing enhancers within the chicken genome. By co-activating promoter and enhancer gRNAs, the study observed higher gene expression, specifically in HBBA and IRF7, compared to the promoter itself. However, no expressed enhancer regions were discovered in the PPARG gene. Notably, the study highlighted the importance of ensuring that co-activated gRNAs exhibit higher expression levels than the promoter gRNA alone to modulate enhancer function effectively. This study provides CRISPR systems that can be used as a gain-of-function platform to validate the functional annotation of the chicken genome and study the molecular mechanisms of genome regulation in poultry genomics research. By evaluating the applicability of CRISPR-based systems in chickens, this study lays the foundation in identifying targets for genetic manipulation and establishing a system for studying gene regulation in chickens. The findings from this research significantly contribute to our understanding of the regulatory mechanisms involved in gene expression within the chicken genome. Furthermore, the study emphasizes the potential for future investigations to leverage these findings to explore the function of enhancers in avian gene regulation.