It is established that for CRISPR-Cas9 applications guide RNAs with 17–20bp long spacer sequences are optimal for accurate target binding and cleavage. In this work we perform cell-free CRISPRa (CRISPR activation) and CRISPRi (CRISPR inhibition) experiments to demonstrate the existence of a complex dependence of CRISPR-Cas9 binding as a function of the spacer length and complementarity. Our results show that significantly truncated or mismatched spacer sequences can form stronger guide–target bonds than the conventional 17–20bp long spacers. To explain this phenomenon, we take into consideration previous structural and single-molecule CRISPR-Cas9 experiments and develop a novel thermodynamic model of CRISPR-Cas9 target recognition.

已经确定，对于 CRISPR-Cas9 应用，具有 17-20bp 长间隔序列的引导 RNA 是准确靶标结合和切割的最佳选择。在这项工作中，我们进行了无细胞 CRISPR（CRISPR 激活）和 CRISPRi（CRISPR 抑制）实验，以证明 CRISPR-Cas9 结合作为间隔长度和互补性的函数存在复杂的依赖性。我们的结果表明，明显截短或错配的间隔序列可以形成比传统的 17-20bp 长的间隔序列更强的引导-目标键。为了解释这一现象，我们考虑了以前的结构和单分子 CRISPR-Cas9 实验，并开发了一种新的 CRISPR-Cas9 目标识别热力学模型。