Edición genética de alta eficiencia mediante inserción dirigida de Cas9 en gen de mantenimiento de pollos

Área de la Ciencia:

• Genomics
• Molecular Biology
• Biotechnology

Sus antecedentes:

• Stable transgene expression is crucial for avian genome engineering but often hindered by epigenetic silencing with viral or piggyBac vectors.
• Existing methods lead to inconsistent, tissue-specific, and diminished expression over time, limiting applications requiring robust, long-term expression.
• Mammalian safe harbor loci (e.g., Rosa26) enable stable expression, but similar strategies are underdeveloped in birds.

Objetivo del estudio:

• To establish a method for stable and ubiquitous transgene expression in chickens by integrating Cas9 into endogenous housekeeping genes.
• To test the hypothesis that targeting safe harbor loci like ACTB and GAPDH can achieve efficient gene editing.
• To provide a robust platform for consistent transgene expression in poultry biotechnology and genome engineering.

Principales métodos:

• Utilized two approaches, 3'-targeted gene insertion and gene tagging, to insert Cas9 and GFP cassettes into chicken DF-1 cells.
• Targeted integration into defined genomic loci, specifically the ACTB and GAPDH housekeeping genes.
• Derived single-cell clones stably expressing Cas9 for downstream genome editing applications.

Principales resultados:

• Both gene insertion methods demonstrated stable transgene expression in chicken DF-1 cells.
• Functional assays confirmed highly efficient Cas9 nuclease activity upon guide RNA delivery.
• Generated single-cell clones with uniform and reproducible genome editing capabilities.

Conclusiones:

• Targeting active housekeeping genes as safe harbor loci mitigates random integration and promoter silencing issues.
• This strategy offers a robust platform for consistent and efficient transgene expression in poultry.
• The developed method advances avian genome engineering by enabling stable and ubiquitous gene expression.