Raman Research Institute, Bangalore, Karnataka, 560080, India.
Sci Rep. 2022 Sep 9;12(1):15243. doi: 10.1038/s41598-022-19485-x.
The CRISPR-associated protein 9 (Cas9) system has proven to be a powerful technology for genome editing in a wide variety of in vivo and in vitro applications. CRISPR-Cas9, when loaded with the guide RNA, cleaves the DNA at the target position as recognized by the guide RNA sequence. For successful application of this technology, it is important to study the biophysical parameters affecting its function. Temperature dependence of the Cas9 binding as well as energetics of product release after cleavage has not been well reported in the literature. In this work, we study the binding properties of Cas9 enzyme to the sequence specific target DNA at a range of temperatures and, surprisingly, find that the Cas9 enzyme, in our study, can find and bind its target DNA with 90 ± 20% efficiency at temperatures as low as 4 °C. Further, we show that the cleaved DNA products remain bound to the Cas9 enzyme strongly and is released from the enzyme only at higher temperatures. Using the gel shift assays, we quantify the rate of Cas9 binding to target DNA to be 0.8 ± 0.2 min at 37 °C. We also tested denaturant (SDS) dependent release of cleaved product which showed a similar release pattern with a dissociation constant of 0.23 ± 0.04 mM. Our results of heat and denaturant dependence on Cas9-DNA binding and release mechanics will provide valuable insights for developing temperature dependent applications of the CRISPR-Cas9 technology.
CRISPR 相关蛋白 9（Cas9）系统已被证明是一种强大的基因组编辑技术，可应用于多种体内和体外应用。当 CRISPR-Cas9 加载向导 RNA 时，会在向导 RNA 序列识别的靶位切割 DNA。为了成功应用这项技术，研究影响其功能的生物物理参数非常重要。文献中尚未很好地报道 Cas9 结合的温度依赖性以及切割后产物释放的能量学。在这项工作中，我们研究了 Cas9 酶在一系列温度下与序列特异性靶 DNA 的结合特性，令人惊讶的是，我们发现 Cas9 酶在我们的研究中可以在低至 4°C 的温度下以 90±20%的效率找到并结合其靶 DNA。此外，我们表明，切割的 DNA 产物仍与 Cas9 酶强烈结合，只有在较高温度下才从酶上释放。使用凝胶迁移分析，我们定量 Cas9 与靶 DNA 结合的速率为 37°C 时为 0.8±0.2 分钟。我们还测试了依赖变性剂（SDS）的切割产物释放，其显示出相似的释放模式，解离常数为 0.23±0.04 mM。我们关于 Cas9-DNA 结合和释放机制对热和变性剂的依赖性的结果将为开发基于温度的 CRISPR-Cas9 技术应用提供有价值的见解。
