Supplement: Acquisition of a Multi-Mode Microplate Reader

项目摘要（母公司奖，GM124413） 该提案将测试CRISPR-Cas12a核酸酶（以前称为CRISPR-Cas12a核酸酶）识别DNA双链体的模型。 "Cpf1"）。我们的目标是了解导致高度DNA歧视的机制基础 在细胞中观察到Cas12a，并且从长远来看，能够更好地设计具有增强的CRISPR变体。 基因组工程的特异性。重复序列（CRISPR） CRISPR相关（Cas）蛋白构成了细菌和古细菌使用的适应性免疫机制 来对抗入侵的病毒和其他移动的遗传元素。在II型CRISPR-Cas9和V型CRISPR-Cas9中， CRISPR-Cas12a系统，一种由CRISPR编码的蛋白激活的单一蛋白组成的效应复合物， 小RNA（s）（crRNA）在特定位点识别并切割双链DNA。开创性的2013年 Cas9可以用工程化的小RNA编程以有效编辑真核基因组的发现 引发了一场基因工程的革命，这场革命仍在迅速展开。Cas12a，首次被鉴定为 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 因此，与Cas9相比，它具有更高的区分力，并且它加工pre-crRNA的能力允许更有效的多重基因组编辑。 有了这些功能，Cas12a在开发更好的基于CRISPR的工具方面具有巨大的潜力。 与Cas9一样，Cas12a中靶向获取的关键步骤是DNA双链体的解旋以形成稳定的DNA双链体。 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杂合体中的错配， 并且Cas12a和Cas9之间的错配容限模式显著不同。然而，机制 引起Cas12a中更高特异性的原因是未知的。 基于现有文献和我们的初步数据，我们提出了Cas12a的两阶段解旋模型。 我们将与Feng Zhang（麻省理工学院和哈佛大学布罗德研究所）的实验室合作，测试该模型 通过探测各种DNA片段的解旋状态，因为Cas12a结合靶双链体并切割每个 这些线这些研究将充分利用我们最近发表的工作， - 定点自旋标记-直接检测Cas9介导的DNA解旋。自旋标记方法将是 结合荧光解旋分析和DNA的详细动力学和热力学分析， 切割和结合。我们希望获得的数据将提供两阶段的明确评估， 解旋模型如果被证明是正确的，该模型将解释Cas12a增强的和独特的错配。 识别模式，并且机制信息将指导基于CRISPR的基因组的进一步开发 编辑技术

Project Summary (parent award, GM124413) 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.