Mitchell Brandon P, Hsu Rohaine V, Medrano Marco A, Zewde Nehemiah T, Narkhede Yogesh B, Palermo Giulia
Department of Bioengineering, University of California, Riverside, Riverside, CA, United States.
Department of Chemistry, University of California, Riverside, Riverside, CA, United States.
Front Mol Biosci. 2020 Mar 17;7:39. doi: 10.3389/fmolb.2020.00039. eCollection 2020.
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.
CRISPR-Cas9是基因组编辑的前沿技术。在该系统中，Cas9蛋白由引导RNA进行编程，以处理与引导RNA匹配的DNA序列，形成RNA:DNA杂交结构。然而，与引导RNA不完全匹配的DNA序列的结合会限制CRISPR-Cas9在基因组编辑中的适用性，从而导致所谓的脱靶效应。在此，利用分子动力学来探究CRISPR-Cas9中RNA:DNA杂交体内DNA碱基对错配的影响。分子模拟显示，相对于原间隔相邻基序（PAM）识别序列，在远端位点的DNA中存在错配碱基对会诱导RNA:DNA杂交体的扩展开放，导致解旋的核酸与蛋白质对应物建立新的相互作用。相反，RNA:DNA杂交体上游的错配碱基对会迅速并入异源双链体，对蛋白质-核酸相互作用的影响较小。因此，PAM远端的错配碱基对会干扰催化性HNH结构域的激活，而完全嵌入RNA:DNA中的错配则不会影响HNH的动力学，并能使其激活以切割DNA。这些发现为有趣的实验证据提供了机制上的理解，即PAM远端错配会阻碍HNH的正常功能，也解释了为什么异源双链体内的错配更能被容忍。这是在理解CRISPR-Cas9脱靶效应方面向前迈出的一步，这鼓励了旨在防止脱靶效应发生的基于新结构的工程学努力。
