A Novel White-to-Blue Colony Formation Assay to Select for Optimized sgRNAs

CRISPR/Cas9-mediated genome editing technology consists of a single-guide RNA (sgRNA), and the Cas9 endonuclease has the potential to treat genetic diseases in most tissues and organisms. In this system, the Cas9 protein can be directed to target genomic DNA sequences as "molecular scissors" with the guidance of sgRNAs. However, the target-specific activities of different sgRNAs are highly variable; thus, it is crucial to search for a simple, quick and economical method to screen for optimized sgRNAs with high target specificity. We have adopted and verified a newly developed white-to-blue colony formation assay to quickly screen for sgRNAs optimized for the EphA2 gene, which is highly expressed in hormone-resistant prostate cancer (PC-3) cells. This assay promises to screen for optimized sgRNAs more simply, rapidly, and efficiently. Our results suggest that the white-to-blue colony formation assay might be a useful screening strategy to quickly select for optimized sgRNAs.