Supplement: Acquisition of a Multi-Mode Microplate Reader

补充：购买多模式酶标仪

基本信息

批准号：
10387736
负责人：
Peter Z Qin
金额：
$ 3.78万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-05 至 2022-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10387736
关键词：
2-Aminopurine Active Sites Archaea Area Award Bacteria Base Pairing Basic Science Binding Biochemical Biological Assay Biophysics CRISPR/Cas technology Cells ChIP-seq Cleaved cell Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Complex DNA DNA Binding DNA analysis Data Development Discrimination Distal Engineering Exposure to Fluorescence Fluorescence Spectroscopy Genes Genome Genome engineering Goals Guide RNA Heteroduplex DNA Human Hybrids In Vitro Institutes Invaded Kinetics Laboratories Literature Mediating Methods Mobile Genetic Elements Modeling Molecular Monitor Mutation Parents Pattern Process Proteins Publications Publishing RNA Reader Role Site Small RNA Specificity Spin Labels Structure System Technology Testing Thermodynamics Variant Virus Work adaptive immunity base biophysical techniques combat design ds-DNA experimental study genome editing genome-wide genome-wide analysis helicase insight mutant next generation nuclease predictive modeling research and development therapeutic development tool

项目摘要

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.

项目总结（家长奖，GM124413）该提案将测试 CRISPR-Cas12a 核酸酶（以前称为 “Cpf1”）的目标是了解导致高度 DNA 歧视的机制基础。观察细胞中的 Cas12a，从长远来看，可以更好地设计具有增强功能的 CRISPR 变体基因组工程的特异性。 CRISPR 相关 (Cas) 蛋白构成细菌和古细菌使用的适应性免疫机制在 II 型 CRISPR-Cas9 和 V 型中对抗入侵病毒和其他可移动遗传元件。 CRISPR-Cas12a 系统，一种由 CRISPR 编码激活的单一蛋白质组成的效应复合物小 RNA (crRNA) 在特定位点识别并切割双链 DNA 突破性的 2013 年。发现 Cas9 可以用工程小 RNA 进行编程，以有效编辑真核基因组 Cas12a 引发了一场仍在迅速展开的基因组工程革命，该革命首次被表征。 2015年底，Cas12a已成功用于基因组编辑，显示出更强的DNA编辑能力。其识别能力优于 Cas9，并且其处理前 crRNA 的能力允许更有效的多重基因组编辑。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，Cas12a 中靶点获取的关键步骤是解开 DNA 双链体以形成稳定的结构。 R 环结构，其中 crRNA 引导片段与 DNA 研究的靶链碱基配对。已经表明 Cas12a 比 Cas9 更严格地辨别 RNA/DNA 杂交体中的错配， Cas12a 和 Cas9 之间的错配容忍模式显着不同，但机制不同。导致 Cas12a 更高特异性的原因尚不清楚。根据现有文献和我们的初步数据，我们提出了 Cas12a 的两阶段展开模型。我们将与张峰实验室（麻省理工学院和哈佛大学博德研究所）合作测试这个模型当 Cas12a 结合目标双链体并切割每个 DNA 片段时，探测各个 DNA 片段的解旋状态这些研究将利用我们最近发表的展示生物物理方法使用的工作。 – 定点自旋标记 – 直接检测 Cas9 介导的 DNA 解旋。结合荧光解旋测定以及详细的 DNA 动力学和热力学分析我们期望获得的数据将为两阶段提供明确的评估。如果证明正确，该模型将解释 Cas12a 的增强且明显的不匹配。判别模式，机制信息将指导基于 CRISPR 的基因组的进一步开发编辑技术。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Experimental Validation of the ALLNOX Program for Studying Protein-Nucleic Acid Complexes.

用于研究蛋白质-核酸复合物的 ALLNOX 程序的实验验证。

DOI：
发表时间：
2019-04-25
期刊：
影响因子：
0
作者：
Ding, Yuan;Kathiresan, Venkatesan;Zhang, Xiaojun;Haworth, Ian S;Qin, Peter Z
通讯作者：
Qin, Peter Z

A DNA unwinding equilibrium serves as a checkpoint for CRISPR-Cas12a target discrimination.

DNA 解旋平衡充当 CRISPR-Cas12a 靶标区分的检查点。

DOI：
发表时间：
2023-09-08
期刊：
影响因子：
14.9
作者：
Singh, Jaideep;Liu, Kevin G;Allen, Aleique;Jiang, Wei;Qin, Peter Z
通讯作者：
Qin, Peter Z

CRISPR-Cas12a Nucleases Bind Flexible DNA Duplexes without RNA/DNA Complementarity.

CRISPR-Cas12a 核酸酶结合灵活的 DNA 双链体，无需 RNA/DNA 互补。

DOI：
发表时间：
2019-10-22
期刊：
影响因子：
4.1
作者：
Jiang, Wei;Singh, Jaideep;Allen, Aleique;Li, Yue;Kathiresan, Venkatesan;Qureshi, Omair;Tangprasertchai, Narin;Zhang, Xiaojun;Parameshwaran, Hari Priya;Rajan, Rakhi;Qin, Peter Z
通讯作者：
Qin, Peter Z

The bridge helix of Cas12a imparts selectivity in cis-DNA cleavage and regulates trans-DNA cleavage.

Cas12a 的桥螺旋赋予顺式 DNA 切割选择性并调节反式 DNA 切割。

DOI：
发表时间：
2021-04
期刊：
影响因子：
3.5
作者：
Parameshwaran, Hari Priya;Babu, Kesavan;Tran, Christine;Guan, Kevin;Allen, Aleique;Kathiresan, Venkatesan;Qin, Peter Z;Rajan, Rakhi
通讯作者：
Rajan, Rakhi