The advent of genome editing platforms such as the CRISPR/Cas9 system ushers an unprecedented speed in the development of new crop varieties that can withstand the agricultural challenges of the 21st century. The CRISPR/Cas9 system depends on the specificity of engineered single guide RNAs (sgRNAs). However, sgRNA design in plants can be challenging due to the multitude of design tools to choose from, many of which use guidelines that are based on animal experiments yet allow the use of plant genomes. Upon choosing sgRNAs, it is also unclear whether an in vitro assay is needed to validate the targeting efficiency of a particular sgRNA before in vivo delivery of the CRISPR/Cas9 system. Here, we demonstrate the in vitro and in vivo activity of four different sgRNAs that we selected based on their ability to target multiple members of the eggplant polyphenol oxidase gene family. Some sgRNAs that have high in vitro cleavage activity did not produce edits in vivo, suggesting that an in vitro assay may not be a reliable basis to predict sgRNAs with highly efficient in vivo cleavage activity. Further analysis of our sgRNAs using other design algorithms suggest that plant-validated criteria such as the presence of necessary secondary structures and appropriate base-pairing may be the reason for the discrepancy between our observed in vitro and in vivo cleavage efficiencies. However, recent reports and our data suggests that there is no guaranteed way to ensure the in vivo cleavage of chosen sgRNAs.

CRISPR/Cas9系统等基因组编辑平台的出现，为能够应对21世纪农业挑战的农作物新品种的开发带来了前所未有的速度。CRISPR/Cas9 系统依赖于工程单引导 RNA (sgRNA) 的特异性。然而，由于有多种设计工具可供选择，植物中的 sgRNA 设计可能具有挑战性，其中许多工具使用基于动物实验的指南，但允许使用植物基因组。在选择 sgRNA 后，还不清楚在体内递送 CRISPR/Cas9 系统之前是否需要进行体外测定来验证特定 sgRNA 的靶向效率。在这里，我们展示了四种不同 sgRNA 的体外和体内活性，我们根据它们靶向茄子多酚氧化酶基因家族多个成员的能力来选择它们。一些具有高体外切割活性的 sgRNA 在体内不会产生编辑，这表明体外测定可能不是预测具有高效体内切割活性的 sgRNA 的可靠基础。使用其他设计算法对我们的 sgRNA 进行进一步分析表明，植物验证的标准（例如必要的二级结构的存在和适当的碱基配对）可能是我们观察到的体外和体内切割效率之间存在差异的原因。然而，最近的报告和我们的数据表明，没有保证的方法可以确保所选 sgRNA 的体内切割。