Investigating mechanisms of DNA unwinding and recognition by a CRISPR-Cas nuclease

项目摘要 这项提议将测试CRISPR-Cas12a核酸酶(以前称为 “cpf1”)。目的是了解导致高度DNA歧视的机制基础 观察细胞中的Cas12a，并从长远来看，使CRISPR变体能够更好地设计 基因组工程的专一性。Clustered-Regularly-Interspaced-Short-Palindromic-Repeats(CRISPR)和 CRISPR相关蛋白(Cas)构成细菌和古菌使用的适应性免疫机制 以对抗入侵的病毒和其他可移动的遗传因素。在类型II CRISPR-CAS9和类型V中 CRISPR-Cas12a系统，由CRISPR编码的单个蛋白激活的效应复合体 小RNA(S)(CrRNA)识别和切割特定位点的双链DNA。开创性的2013年 发现Cas9可以用工程小RNA编程来有效编辑真核基因组 引发了一场仍在迅速展开的基因组工程革命。Cas12a，它首先被描述为 2015年底，已成功用于基因组编辑。Cas12a显示出更强的DNA识别能力 其处理前crRNA的能力使多重基因组编辑更有效率。 凭借这些功能，Cas12a具有开发更好的基于CRISPR的工具的巨大潜力。 与Cas9一样，在Cas12a中获得靶的关键步骤是解开DNA双链以形成稳定的 R-环结构，其中crRNA引导片段与DNA的靶链碱基配对。研究 已经表明，Cas12a比Cas9更严格地区分RNA/DNA杂交中的错配， CAS12a和CAS9之间的失配容差模式存在显著差异。然而，这种机制 这导致了Cas12a中较高的特异性是未知的。 基于现有的文献和我们的初步数据，我们提出了Cas12a的两阶段解锁模型。 我们将与张峰(麻省理工学院和哈佛大学)的实验室合作，测试该模型 通过探测当Cas12a结合目标双链并切割每个DNA片段时不同DNA片段的解离状态 绳索。这些研究将利用我们最近发表的工作来演示生物物理方法的使用 -定点定向自旋标记-直接检测Cas9介导的DNA解离。自旋标记方法将是 结合荧光解旋分析和详细的DNA动力学和热力学分析 卵裂和捆绑。我们期望所获得的数据将对这两个阶段作出明确的评估。 展开模型。如果被证明是正确的，该模型将解释Cas12a增强的和明显的不匹配 识别模式和机制信息将指导基于CRISPR的基因组的进一步发展 编辑技术。

Project Summary This proposal will test a model of DNA duplex recognition by the CRISPR-Cas12a nuclease (previously called “Cpf1”). The goal is to understand the mechanistic basis leading to the high degree of DNA discrimination observed for Cas12a in the cell, and, in the long term, to enable better design of CRISPR variants with enhanced specificity for genome engineering. Clustered-Regularly-Interspaced-Short-Palindromic-Repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute an adaptive immunity mechanism used by bacteria and archaea to combat invading viruses and other mobile genetic elements. In both the type II CRISPR-Cas9 and the type V CRISPR-Cas12a systems, an effector complex comprised of a single protein activated by CRISPR-encoded small RNA(s) (crRNA) recognizes and cleaves double-stranded DNAs at specific sites. The groundbreaking 2013 discovery that Cas9 can be programmed with engineered small RNAs to efficiently edit eukaryotic genomes sparked a revolution in genome engineering that is still rapidly unfolding. Cas12a, which was first characterized at the end of 2015, has been successfully used for genome editing. Cas12a shows stronger capability for DNA discrimination than Cas9, and its ability to process the pre-crRNA allows more efficient multiplex genome editing. With these features, Cas12a holds great potentials for development of better CRISPR-based tools. As with Cas9, a key step in target acquisition in Cas12a is the unwinding of the DNA duplex to form a stable R-loop structure, in which the crRNA guide-segment is base-paired with the target-strand of the DNA. Studies have shown that Cas12a more stringently discriminates against mismatches in the RNA/DNA hybrid than Cas9, and the mismatch tolerance patterns significantly differ between Cas12a and Cas9. However, the mechanism that gives rise to the higher specificity in Cas12a is unknown. Based on available literature and our preliminary data, we propose a two-stage unwinding model for Cas12a. In collaboration with the laboratory of Feng Zhang (Broad Institute of MIT and Harvard), we will test this model by probing the unwinding state of various DNA segments as Cas12a binds a target duplex and cleaves each of the strands. The studies will leverage our recently published work demonstrating the use of a biophysical method – site-directed spin labeling – to directly detect Cas9-mediated DNA unwinding. The spin labeling method will be combined with a fluorescence unwinding assay and detailed kinetic and thermodynamic analyses of DNA cleavage and binding. We expect that data obtained will provide a definitive assessment of the two-stage unwinding model. If proven correct, the model will account for Cas12a’s enhanced and distinct mismatch discrimination pattern, and the mechanistic information will guide further development of CRISPR-based genome editing technology.