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.

项目总结/摘要 CRISPR（规则间隔短回文重复序列）系统的发现导致了 创造性的新应用正在改变科学和医学。然而，新的快速发现 CRISPR系统超越了我们对它们在抗噬菌体防御中的生物学作用的理解， 开发新的应用。为了获得对新病毒的免疫力，CRISPR相关蛋白（Cas 1 和Cas 2）在CRISPR基因座的“前导端”，在CRISPR基因座的“前导端”附近整合噬菌体DNA的片段（“间隔区”）。 转录起始位点。但是Cas 1 -2如何识别CRISPR的前导端仍然知之甚少。 接下来，CRISPR位点被转录并加工成“向导RNA”， 复合物（即，CSM复合物）。在检测病毒RNA时，Csm复合物使环状寡核苷酸 调节CRISPR适应的信使和对噬菌体防御至关重要的核酸酶。但生物学角色 这些免疫效应子的研究还不充分。我对CRISPR的初步生物信息学分析 前导序列（上游DNA）揭示了亚型和微生物特异性基序。这些保守的基序被发现 在相对于前导序列-CRISPR连接的位置的紧密分布中，并且位置变体被移位了一个或多个位置。 DNA的螺旋转动，将这些基序保留在DNA的同一“面上”。在目标1中， 将确定生物化学捕获的CRISPR整合复合物的结构，以确定 前导基序和宿主蛋白调节整合。在目标2中，我将确定一种生物化学和结构 我预测这些免疫蛋白会被CRISPR产生的核苷酸信使激活。 这份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： 生物学培训，ii）指导、沟通和资助写作培训，iii）新型CRISPR的鉴定 适应调节基序和，iv）鉴定新的免疫效应物。我会接受正规的冷冻电镜检查 课程由共同顾问劳伦斯博士在蒙大拿州立大学（MSU）的冷冻EM设施提供， Lander博士，辅以NIH国家冷冻EM访问中心的课程， 训练我将进一步发展我的指导，沟通，并通过课程写作技巧 由密歇根州立大学的卓越教师中心和ASBMB的UE 5组件提供。我招募了一个 指导和咨询委员会的六名科学家在CRISPR生物学互补的专业知识， 基于CRISPR的应用开发，cryo-EM方法学，病毒学和传染病- 在我追求研究和职业目标的过程中，它对我有着重要的指导意义。该项目为我提供了 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物化学数据， 将作为我独立实验室跨学科研究CRISPR生物学及其 应用.在那里，我的目标是成为下一代代表性不足的少数民族科学家的导师。