Structural Basis of Programmable DNA-Insertion via Cryo-EM Studies of CRISPR-Associated TnsC

通过冷冻电镜研究 CRISPR 相关 TnsC 的可编程 DNA 插入的结构基础

• 批准号: 10797749
• 负责人: Elizabeth Kellogg
• 金额: $ 20.94万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2023-08-10
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10797749
• 关键词: Administrative Supplement; Award; CRISPR-associated transposons; Clustered Regularly Interspaced Short Palindromic Repeats; Cryoelectron Microscopy; DNA; DNA Binding; DNA Transposons; Data Set; Disparate; Equipment; Funding; Guide RNA; Image; Nucleoproteins; Parents; Process; RNA Sequences; Research; Site; Structure; Time; Transposase; United States National Institutes of Health; computing resources; experience; interest; programs

The current major focus of the parent award (R01) is to reveal how CRISPR-associated transposons (CAST) exquisitely regulate their insertion activity, which is dependent on a guide- RNA sequence. Specifically, we are interested in how CAST elements: 1. choose their target- sites and 2. how transposase catalytic activity is properly activated at the target site. We believe that the physical arrangement of the CRISPR and transposase functional modules is the key to describe how disparate functionality in guide-RNA recognition and target-DNA binding are integrated and regulated. To accomplish this, cryo-electron microscopy (Cryo-EM) is used to obtain large imaging datasets of megadalton nucleoprotein assemblies, which must then be analyzed and processed using a computationally intensive workflow. The Kellogg lab maintains its own local server, equipped with 4 A100, which are suitable for analyzing one dataset at a time. However, the large size of the datasets currently imaged necessitate an expansion of current computing resources. To this end, we request an administrative equipment supplement to allow us to expand computing by 3-fold through the purchase of an additional server with 8 A100 GPUs. This will allows us to keep up with current computational bottlenecks experienced throughout the NIH funded research program.

目前家长奖（R01）的主要焦点是揭示CRISPR如何与 转座子（CAST）精确地调节它们的插入活性，这取决于引导- RNA 序列。具体来说，我们感兴趣的是 CAST 元素如何： 1. 选择他们的目标 - 2. 转座酶催化活性如何在靶位点正确激活。我们相信 CRISPR和转座酶功能模块的物理排列是关键 描述引导 RNA 识别和目标 DNA 结合的不同功能 整合和监管。为了实现这一目标，使用冷冻电子显微镜 (Cryo-EM) 获得百万道尔顿核蛋白组装体的大型成像数据集，然后必须将其 使用计算密集型工作流程进行分析和处理。凯洛格实验室坚持 拥有自己的本地服务器，配备4台A100，适合同时分析一个数据集 时间。然而，目前成像的数据集规模很大，需要扩展 当前的计算资源。为此，我们要求补充行政设备 通过购买额外的 8 台服务器，我们可以将计算能力扩展 3 倍 A100 GPU。这将使我们能够跟上当前遇到的计算瓶颈 整个 NIH 资助的研究项目。