Efficient Genome Editing in Chicken DF-1 Cells Using the CRISPR/Cas9 System.

Area of Science:

• Genomics
• Molecular Biology
• Gene Editing

Background:

• Genome engineering offers precise DNA modification capabilities.
• CRISPR/Cas9 is a powerful tool for targeting specific genomic loci across various organisms.
• Limited data exists on CRISPR/Cas9 applications in avian species.

Purpose of the Study:

• To investigate the efficacy of CRISPR/Cas9 for inducing mutations in chicken DF-1 cells.
• To evaluate the utility of a surrogate reporter system for enhancing gene editing efficiency in chickens.
• To assess potential off-target mutations in CRISPR/Cas9-mediated chicken genome editing.

Main Methods:

• CRISPR/Cas9 system was utilized to target PPAR-γ, ATP5E, and OVA genes in chicken DF-1 cells.
• T7E1 assays were performed to assess mutation rates before and after enrichment.
• A surrogate reporter system involving the Puro(R) gene was employed to enrich modified cells.
• Sequence analysis confirmed mutation efficiency, and potential off-target sites were analyzed.

Main Results:

• Initial mutation rates for PPAR-γ, ATP5E, and OVA genes were low (0.75%, 0.5%, 3.0%).
• Enrichment using the surrogate reporter system dramatically increased mutation rates to over 60% (T7E1 assay).
• Subsequent sequence analysis confirmed high mutation efficiencies of 94.7%, 95%, and 95% for the targeted genes.
• No detectable off-target mutations were observed at three potential off-target sites.

Conclusions:

• The CRISPR/Cas9 system is effective for genome editing in chicken DF-1 cells.
• Surrogate reporter systems significantly enhance the efficiency of CRISPR/Cas9-mediated gene editing in chickens.
• CRISPR/Cas9 demonstrates high specificity with no detectable off-target effects in this study, making it a robust tool for chicken genome modification.