Key result
Using a surrogate reporter system with the Puro(R) gene increased CRISPR/Cas9 mutation efficiency in chicken DF-1 cells from ≤3.0% to up to 95% without detectable off-target mutations.
The CRISPR/Cas9 system, particularly when combined with a surrogate reporter system for enrichment, is a highly efficient tool for targeted genome editing in chicken cells.
Enhances CRISPR efficiency in chicken cells; leaves open translation to primary tissues or in vivo models.
In recent years, genome engineering technology has provided unprecedented opportunities for site-specific modification of biological genomes. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 is one such means that can target a specific genome locus. It has been applied in human cells and many other organisms. Meanwhile, to efficiently enrich targeted cells, several surrogate systems have also been developed. However, very limited information exists on the application of CRISPR/Cas9 in chickens. In this study, we employed the CRISPR/Cas9 system to induce mutations in the peroxisome proliferator-activated receptor-γ (PPAR-γ), ATP synthase epsilon subunit (ATP5E), and ovalbumin (OVA) genes in chicken DF-1 cells. The results of T7E1 assays showed that the mutation rate at the three different loci was 0.75%, 0.5%, and 3.0%, respectively. In order to improve the mutation efficiency, we used the Puro(R) gene for efficient enrichment of genetically modified cells with the surrogate reporter system. The mutation rate, as assessed via the T7E1 assay, increased to 60.7%, 61.3%, and 47.3%, and subsequent sequence analysis showed that the mutation efficiency increased to 94.7%, 95%, and 95%, respectively. In addition, there were no detectable off-target mutations in three potential off-target sites using the T7E1 assay. As noted above, the CRISPR/Cas9 system is a robust tool for chicken genome editing.
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Bai et al. (2016) studied this question. CRISPR/Cas9 system with Puro(R) gene surrogate reporter system vs. CRISPR/Cas9 system without enrichment was evaluated on Mutation rate at PPAR-γ, ATP5E, and OVA genes. Using a surrogate reporter system with the Puro(R) gene increased CRISPR/Cas9 mutation efficiency in chicken DF-1 cells from ≤3.0% to up to 95% without detectable off-target mutations.
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