CRISPR/Cas9-mediated gene targeting for robust transgene expression in chickens
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This thesis explores the development and implementation of a targeted gene integration strategy using CRISPR/Cas9 to achieve robust and consistent transgene expression in chickens. Stable transgene expression remains a critical challenge in avian genetic engineering due to the limitations of random integration approaches, such as piggyBac transposition and viral transduction, which are prone to epigenetic silencing and positional effects. To address this, we investigated the use of two ubiquitously expressed housekeeping genes—GAPDH and ACTB—as potential genomic safe harbor loci for precise transgene insertion in chicken fibroblast (DF-1) cells and primordial germ cells (PGCs). Guide RNAs were designed to target the 3′ coding and intronic regions of these genes, enabling Cas9-mediated double-strand breaks followed by non-homologous end joining (NHEJ)-driven knock-in of reporter constructs. Editing efficiency and indel formation were evaluated via T7E1 assays and Sanger sequencing, with GAPDH demonstrating superior integration rates and consistent GFP expression. Knock-in cell lines were further validated by functional Cas9 activity, and successful targeted integration in PGCs suggests potential for germline transmission. In addition to presenting experimental results, this thesis includes a comprehensive literature review on the evolution of genome editing technologies in chickens, challenges in transgene silencing, and the rationale for targeting housekeeping genes. Collectively, this work establishes a foundation for the development of reliable, site-specific transgene expression systems in poultry, with implications for agricultural biotechnology, pharmaceutical protein production, and basic research.
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M.S.