Effect of Cas9 Protein on the Seed-Target Base Pair of the sgRNA/DNA Hybrid Duplex.
Effect of Cas9 Protein on the Seed-Target Base Pair of the sgRNA/DNA Hybrid Duplex.
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DOI：
10.1021/acs.jpcb.3c00997
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发表时间：
2023-05
期刊：
影响因子：
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通讯作者：
Ting Yu;Taigang Liu;Yujie Wang;Xuanlin Zhao;Wenbing Zhang
中科院分区：
文献类型：
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作者：
Ting Yu;Taigang Liu;Yujie Wang;Xuanlin Zhao;Wenbing Zhang
CRISPR（成簇的规则间隔短回文重复序列）/CRISPR相关蛋白（Cas9）已被广泛用于基因编辑。并非所有的向导RNA都可以有效地切割DNA，这仍然是CRISPR/Cas9介导的基因组工程的主要挑战。因此，了解Cas9复合物如何通过碱基配对成功有效地识别特定的功能靶标对此类应用具有重大意义。引导RNA 3'端的10-nt种子序列对于靶标识别和切割至关重要。在这里，我们通过拉伸分子动力学模拟，研究了种子碱基和靶DNA碱基与Cas9蛋白结合-解离过程的热力学和动力学。结果表明，在Cas9蛋白存在下，种子碱基与靶标的结合-解离的焓变和熵变小于没有Cas9蛋白的那些。与蛋白质缔合后熵罚的减少是由于种子碱基以A型螺旋的预组织，而焓变的减少是由于带正电荷的通道与带负电荷的靶DNA的静电吸引。在Cas9蛋白存在下，来自熵损失的结合屏障和由碱基对破坏引起的解离屏障低于没有蛋白的那些，这表明种子区域通过加速结合速率和快速从错误的靶标解离来有效地搜索正确的靶标是至关重要的。
CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associated protein (Cas9) has been widely used for gene editing. Not all guide RNAs can cleave the DNA efficiently remains a major challenge to CRISPR/Cas9-mediated genome engineering. Therefore, understanding how the Cas9 complex successfully and efficiently identifies specific functional targets through base-pairing has great implications for such applications. The 10-nt seed sequence at the 3' end of the guide RNA is critical to target recognition and cleavage. Here, through stretching molecular dynamics simulation, we studied the thermodynamics and kinetics of the binding-dissociation process of the seed base and the target DNA base with the Cas9 protein. The results showed that in the presence of Cas9 protein, the enthalpy change and entropy change in binding-dissociation of the seed base with the target are smaller than those without the Cas9 protein. The reduction of entropy penalty upon association with the protein resulted from the pre-organization of the seed base in an A-form helix, and the reduction of enthalpy change was due to the electrostatic attraction of the positively charged channel with the negative target DNA. The binding barrier coming from the entropy loss and the dissociation barrier resulting from the destruction of the base pair in the presence of Cas9 protein were lower than those without protein, which indicates that the seed region is crucial for efficiently searching the correct target by accelerating the binding rate and dissociating fast from the wrong target.