Spontaneous Embedding of DNA Mismatches Within the RNA:DNA Hybrid of CRISPR-Cas9
Spontaneous Embedding of DNA Mismatches Within the RNA:DNA Hybrid of CRISPR-Cas9
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DOI：
10.3389/fmolb.2020.00039
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发表时间：
2020-03-17
影响因子：
5
通讯作者：
Palermo, Giulia
中科院分区：
文献类型：
--
作者：
Mitchell, Brandon P.;Hsu, Rohaine, V;Palermo, Giulia
CRISPR-Cas9是编辑基因组的最前沿技术。在该系统中，Cas9蛋白用向导RNA编程以加工与向导RNA匹配的DNA序列，从而形成RNA：DNA杂交结构。然而，与指导RNA不完全匹配的DNA序列的结合会限制CRISPR-Cas9用于基因组编辑的适用性，导致所谓的脱靶效应。在这里，分子动力学用于探测CRISPR-Cas9中RNA：DNA杂交体内DNA碱基对错配的影响。分子模拟显示，在DNA中的错配对的存在下，在远端站点相对于Protospacer相邻基序（PAM）的识别序列诱导的RNA：DNA杂交延长开放，导致新的相互作用建立的解绕核酸和蛋白质对应物。相反，RNA：DNA杂交体上游的错配对被迅速并入异源双链体内，对蛋白质-核酸相互作用的影响很小。因此，PAM远端的错配对会干扰催化HNH结构域的激活，而完全嵌入RNA中的错配：DNA不会影响HNH动力学，并使其激活以切割DNA。这些发现为有趣的实验证据提供了一种机制上的理解，即PAM远端错配阻碍了HNH的正常功能，也解释了为什么异源双链体内的错配更容易被容忍。这在理解CRISPR-Cas9中的脱靶效应方面向前迈出了一步，这鼓励了旨在防止脱靶效应发生的基于新结构的工程努力。
CRISPR-Cas9 is the forefront technology for editing the genome. In this system, the Cas9 protein is programmed with guide RNAs to process DNA sequences that match the guide RNA forming an RNA:DNA hybrid structure. However, the binding of DNA sequences that do not fully match the guide RNA can limit the applicability of CRISPR-Cas9 for genome editing, resulting in the so-called off-target effects. Here, molecular dynamics is used to probe the effect of DNA base pair mismatches within the RNA:DNA hybrid in CRISPR-Cas9. Molecular simulations revealed that the presence of mismatched pairs in the DNA at distal sites with respect to the Protospacer Adjacent Motif (PAM) recognition sequence induces an extended opening of the RNA:DNA hybrid, leading to novel interactions established by the unwound nucleic acids and the protein counterpart. On the contrary, mismatched pairs upstream of the RNA:DNA hybrid are rapidly incorporated within the heteroduplex, with minor effect on the protein-nucleic acid interactions. As a result, mismatched pairs at PAM distal ends interfere with the activation of the catalytic HNH domain, while mismatches fully embedded in the RNA:DNA do not affect the HNH dynamics and enable its activation to cleave the DNA. These findings provide a mechanistic understanding to the intriguing experimental evidence that PAM distal mismatches hamper a proper function of HNH, explaining also why mismatches within the heteroduplex are much more tolerated. This constitutes a step forward in understanding off-target effects in CRISPR-Cas9, which encourages novel structure-based engineering efforts aimed at preventing the onset of off-target effects.