Necessity for Validation of Effectiveness of Selected Guide RNA In Silico for Application of CRISPR/Cas9.

Selection of guide RNA (gRNA) is important to increase the efficiency of gene editing in the CRISPR/Cas9 system. Due to the variation in actual efficiency of insertion/deletion (indel) mutation among selected gRNAs in silico, reliable methods for validation of efficiency of gRNA need to be developed. Three gRNAs with high on-target scores (72.0 for target 1, 65.4 for target 2, and 62.9 for target 3) were designed to target the quail retinol binding protein 7 (qRbp7) gene, and indel efficiencies were predicted by the Sanger sequencing and Inference of CRISPR Edits (ICE) analysis of sorted cell populations receiving the CRISPR/Cas9 vector. Unlike the order of on-target scores among 3 gRNAs, predicted rates of indel mutations were highest in gRNA2, intermediate in gRNA1, and lowest in gRNA3. This was confirmed by actual indel mutation rates, 51.8% in gRNA2, 31% in gRNA1, and 12.9% in gRNA3, which were calculated by sequencing individual allele cloned into a vector. These data showed a rapid and reliable method for estimation of the efficiency of selected gRNAs, providing a critical necessary step for successful gene editing for further applications.