Complex dependence of CRISPR-Cas9 binding strength on guide RNA spacer lengths
Complex dependence of CRISPR-Cas9 binding strength on guide RNA spacer lengths
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DOI：
10.1088/1478-3975/ac091e
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发表时间：
2021-09-01
期刊：
影响因子：
2
通讯作者：
Noireaux, Vincent
中科院分区：
文献类型：
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作者：
Khakimzhan, Aset;Garenne, David;Noireaux, Vincent
已经确定，对于CRISPR-Cas9应用，具有17-20bp长间隔序列的引导RNA对于准确的靶标结合和切割是最佳的。在这项工作中，我们进行了无细胞CRISPRa(CRISPR激活)和CRISPRi(CRISPR抑制)实验，以证明CRISPR-Cas9结合作为间隔子长度和互补性的函数存在复杂依赖关系。我们的结果表明，显著截断或错配的间隔区序列可以形成比传统的17-20个碱基长的间隔区更强的导向靶键。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，并发展了一个新的CRISPR-Cas9目标识别的热力学模型。
It is established that for CRISPR-Cas9 applications guide RNAs with 17-20 bp 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 17-20 bp 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.