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.

家长奖(R01)目前的主要重点是揭示CRISPR如何与 转座子(CAST)精细地调节它们的插入活性，这种插入活性依赖于一个向导- RNA序列。具体地说，我们感兴趣的是如何铸造元素：1.选择他们的目标- 2.转座酶催化活性如何在目标部位被适当地激活。我们相信 CRISPR和转座酶功能模块的物理排列是 描述GUIDE-RNA识别和靶DNA结合中的不同功能是如何 整合和规范。为了做到这一点，冷冻电子显微镜(Cryo-EM)被用于 获得百万吨核蛋白组件的大型成像数据集，然后必须 使用计算密集型工作流进行分析和处理。凯洛格实验室坚持认为 它自己的本地服务器，配备了4个A100，适合一次分析一个数据集 时间到了。然而，当前成像的数据集的大小需要扩展 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，我们可以将计算扩展到原来的3倍 A100图形处理器。这将使我们能够跟上当前遇到的计算瓶颈 在整个NIH资助的研究项目中。