The therapeutic application of CRISPR-Cas9 is limited due to its off-target activity. To have a better understanding of this off-target effect, we focused on its mismatch-prone PAM distal end. The off-target activity of SpCas9 depends directly on the nature of mismatches, which in turn results in deviation of the active site of SpCas9 due to structural instability in the RNA-DNA duplex strand. In order to test the hypothesis, we designed an array of mismatched target sites at the PAM distal end and performed and cell line-based experiments, which showed a strong correlation for Cas9 activity. We found that target sites having multiple mismatches in the 18th to 15th position upstream of the PAM showed no to little activity. For further mechanistic validation, Molecular Dynamics simulations were performed, which revealed that certain mismatches showed elevated root mean square deviation values that can be attributed to conformational instability within the RNA-DNA duplex. Therefore, for successful prediction of the off-target effect of SpCas9, along with complementation-derived energy, the RNA-DNA duplex stability should be taken into account.

中文翻译：

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SpCas9对PAM远端失配耐受性的机制研究


CRISPR-Cas9的治疗应用由于其脱靶活性而受到限制。为了更好地理解这种脱靶效应，我们重点关注其容易失配的 PAM 远端。 SpCas9的脱靶活性直接取决于错配的性质，进而导致由于RNA-DNA双链体结构不稳定而导致SpCas9活性位点的偏差。为了验证这一假设，我们在 PAM 远端设计了一系列不匹配的靶位点，并进行了基于细胞系的实验，结果表明 Cas9 活性具有很强的相关性。我们发现，在 PAM 上游第 18 至 15 位具有多个错配的靶位点没有表现出活性。为了进一步进行机械验证，进行了分子动力学模拟，结果表明某些不匹配表现出均方根偏差值升高，这可归因于 RNA-DNA 双链体内的构象不稳定性。因此，为了成功预测 SpCas9 的脱靶效应以及互补产生的能量，应考虑 RNA-DNA 双链体的稳定性。