ABSTRACT

RNAi is a potent technique for the knockdown of target genes. However, its potential off-target effects limit the widespread applications in both reverse genetic analysis and genetic manipulation. Previous efforts have uncovered rules underlying specificity of siRNA-based silencing, which has broad applications in humans, but the basis for specificity of dsRNAs, which are better suited for use as insecticides, is poorly understood. Here, we investigated the rules governing dsRNA specificity. Mutational analyses showed that dsRNAs with >80% sequence identity with target genes triggered RNAi efficiently. dsRNAs with ≥16 bp segments of perfectly matched sequence or >26 bp segments of almost perfectly matched sequence with one or two mismatches scarcely distributed (single mismatches inserted between ≥5 bp matching segments or mismatched couplets inserted between ≥8 bp matching segments) also able to trigger RNAi. Using these parameters to predict off-target risk, dsRNAs can be designed to optimize specificity and efficiency, paving the way to the widespread, rational application of RNAi in pest control.

摘要

RNAi 是一种有效的靶基因敲除技术。然而，其潜在的脱靶效应限制了在反向遗传分析和遗传操作中的广泛应用。先前的努力已经揭示了基于 siRNA 的沉默特异性的潜在规则，这在人类中具有广泛的应用，但更适合用作杀虫剂的 dsRNA 特异性的基础却​​知之甚少。在这里，我们调查了管理 dsRNA 特异性的规则。突变分析表明，与靶基因具有>80% 序列同一性的 dsRNA 有效地触发了 RNAi。具有完全匹配序列的 ≥16 bp 片段的 dsRNA 或 > 几乎完全匹配序列的 26 bp 片段，几乎不分布一或两个错配（插入≥5 bp 匹配片段之间的单个错配或插入≥8 bp 匹配片段之间的错配对）也能够触发 RNAi。使用这些参数来预测脱靶风险，可以设计 dsRNA 以优化特异性和效率，为 RNAi 在害虫防治中的广泛、合理应用铺平道路。