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.

向导 RNA (gRNA) 的选择对于提高 CRISPR/Cas9 系统中基因编辑的效率非常重要。由于计算机中选定的 gRNA 插入/缺失 (indel) 突变的实际效率存在差异，因此需要开发可靠的方法来验证 gRNA 的效率。设计了三种具有高在靶得分（靶标 1 为 72.0、靶标 2 为 65.4、靶标 3 为 62.9）的 gRNA 来靶向鹌鹑视黄醇结合蛋白 7 (qRbp7) 基因，并通过 Sanger 测序和预测预测了插入缺失效率。对接受 CRISPR/Cas9 载体的分选细胞群进行 CRISPR 编辑推断 (ICE) 分析。与 3 个 gRNA 的中靶得分顺序不同，预测的 indel 突变率在 gRNA2 中最高，在 gRNA1 中居中，在 gRNA3 中最低。实际插入/缺失突变率证实了这一点，gRNA2 为 51.8%，gRNA1 为 31%，gRNA3 为 12.9%，这些突变率是通过对克隆到载体中的单个等位基因进行测序计算得出的。这些数据显示了一种快速可靠的方法来估计所选 gRNA 的效率，为成功的基因编辑进一步应用提供了关键的必要步骤。