Mechanisms of CRISPR-mediated immunity and applications beyond editing

CRISPR介导的免疫机制和编辑以外的应用

• 批准号: 10515419
• 负责人: Andrew Santiago-Frangos
• 金额: $ 10万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10515419
• 关键词: Adaptive Immune System; Advisory Committees; Archaea; Area; Award; Bacteria; Bacteriophages; Binding; Biochemical; Biochemistry; Bioinformatics; Biological; Biology; Biotechnology; Cell physiology; Cellular Morphology; Cluster Analysis; Clustered Regularly Interspaced Short Palindromic Repeats; Communicable Diseases; Communication; Complex; Cryoelectron Microscopy; Cyclic Nucleotides; DNA; DNA Integration; DNA Sequence; Data; Deaminase; Development; Devices; Diagnostic; Face; Faculty; Foundations; Future; Genetic; Genome; Goals; Grant; Guide RNA; Immune; Immune response; Immune signaling; Immunity; Industry; Integration Host Factors; Interdisciplinary Study; Knowledge; Letters; Life; Ligands; Mediating; Medicine; Mentors; Methodology; Methods; Microbe; Microscope; Mission; Modeling; Montana; Nucleotides; Oligonucleotides; Peptide Hydrolases; Periodicity; Positioning Attribute; Process; Proteins; Pseudomonas; Pseudomonas aeruginosa; Public Health; RNA Phages; Research; Resolution; Retrieval; Role; Science; Scientist; Signal Pathway; Signaling Molecule; Structure; System; Technology; Testing; Thermus thermophilus; Training; Transcription Initiation Site; Trees; Underrepresented Minority; United States National Institutes of Health; Universities; Variant; Viral; Writing; antimicrobial; base; career; genetic information; insight; interest; microbial; minority scientist; next generation; novel; nuclease; preservation; programs; recruit; skills; structural biology; viral RNA; viral detection; virology

PROJECT SUMMARY/ABSTRACT The discovery of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems led to creative new applications that are transforming science and medicine. However, the rapid discovery of new CRISPR systems outpaces our understanding of their biological roles in anti-phage defense and their development for novel applications. To acquire immunity to new phages, CRISPR-associated proteins (Cas1 and Cas2) integrate fragments of phage DNA ("spacers") at the "leader-end" of the CRISPR locus, near the transcription start site. But how Cas1-2 recognizes the leader-end of the CRISPR remains poorly understood. Next, the CRISPR locus is transcribed and processed into "guide RNAs" that are loaded into surveillance complexes (i.e., Csm complex). Upon sensing viral RNA, the Csm complex makes cyclic oligonucleotide messengers that regulate CRISPR adaptation and nucleases critical for phage defense. But the biological roles of many of these immune effectors remain understudied. My preliminary bioinformatics analysis of CRISPR leaders (upstream DNA) has revealed subtype- and microbe-specific motifs. These conserved motifs are found in a tight distribution of positions relative to the leader-CRISPR junction, and positional variants are shifted by a helical turn of DNA that would preserve the presentation of these motifs on the same "face" of DNA. In Aim 1, I will determine the structures of biochemically trapped CRISPR integration complexes to determine the roles of leader motifs and host proteins in regulating integration. In Aim 2, I will determine a biochemical and structural understanding of immune proteins that I predict to be activated by CRISPR-generated nucleotide messengers. This tailored application will support my transition to an independent academic position through: i) Structural biology training, ii) Mentoring, communication, and grant writing training, iii) Identification of novel CRISPR adaptation regulatory motifs and, iv) Identification of novel immune effectors. I will take formal cryo-EM coursework provided at Montana State 'University's (MSUs) cryo-EM facility by co-advisor Dr. Lawrence and co-mentor Dr. Lander, supplemented by coursework at NIH's National Center for Cryo-EM Access and Training. I will further develop my mentoring, communication, and grant writing skills through coursework provided by 'MSU's Center for Faculty excellence and by ASBMB's UE5 component. I have recruited a mentoring and advisory committee of six scientists with complementary expertise in CRISPR biology, the development of CRISPR-based applications, cryo-EM methodology, virology, and infectious diseases - relevant to guiding me as I pursue my research and career goals. The proposed project provides me with training in cryo-EM structural biology. It provides me with foundational bioinformatic and biochemical data that will serve as a springboard for my independent lab's interdisciplinary research on CRISPR biology and its applications. There, I aim to serve as a mentor for the next generation of underrepresented minority scientists.

项目概要/摘要 CRISPR（成簇规则间隔短回文重复）系统的发现导致 正在改变科学和医学的创造性新应用。然而，新的快速发现 CRISPR 系统超出了我们对其在抗噬菌体防御中的生物学作用及其作用的理解。 新颖应用的开发。为了获得对新噬菌体、CRISPR 相关蛋白 (Cas1 和 Cas2) 将噬菌体 DNA 片段（“间隔区”）整合到 CRISPR 位点的“前导端”，靠近 转录起始位点。但 Cas1-2 如何识别 CRISPR 的引导端仍知之甚少。 接下来，CRISPR 位点被转录并加工成“引导 RNA”，并加载到监视中 复合物（即 Csm 复合物）。在检测到病毒 RNA 后，Csm 复合物会产生环状寡核苷酸 调节 CRISPR 适应的信使和对噬菌体防御至关重要的核酸酶。但生物学作用 其中许多免疫效应物仍未得到充分研究。我对CRISPR的初步生物信息学分析 领导者（上游 DNA）揭示了亚型和微生物特异性基序。发现这些保守的基序 相对于前导-CRISPR连接点的位置分布紧密，并且位置变异通过 DNA 的螺旋转动将保留这些图案在 DNA 的同一“面”上的呈现。在目标 1 中，我 将确定生化捕获的 CRISPR 整合复合物的结构，以确定 前导基序和宿主蛋白在调节整合中的作用。在目标 2 中，我将确定生化和结构 了解我预测会被 CRISPR 生成的核苷酸信使激活的免疫蛋白。 这个量身定制的申请将通过以下方式支持我向独立学术职位的过渡： i) 结构 生物学培训，ii) 指导、沟通和资助写作培训，iii) 新型 CRISPR 的鉴定 适应调节基序，以及，iv) 新型免疫效应物的鉴定。我将采取正式的冷冻电镜 蒙大拿州立大学 (MSU) 冷冻电镜设施由联合顾问 Lawrence 博士和 联合导师 Lander 博士，并辅以 NIH 国家冷冻电镜中心的课程作业和 训练。我将通过课程进一步发展我的指导、沟通和资助写作技能 由“MSU 卓越教师中心”和 ASBMB 的 UE5 组件提供。我已经招募了一个 由六位在 CRISPR 生物学领域具有互补专业知识的科学家组成的指导和咨询委员会 基于 CRISPR 的应用、冷冻电镜方法、病毒学和传染病的开发 - 与我追求研究和职业目标相关的指导。拟议的项目为我提供了 冷冻电镜结构生物学培训。它为我提供了基础的生物信息学和生化数据 将作为我的独立实验室 CRISPR 生物学及其相关跨学科研究的跳板 应用程序。在那里，我的目标是成为下一代代表性不足的少数族裔科学家的导师。