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.

项目摘要/摘要 CRISPR(簇状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，新技术的快速发现 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和它们的 开发新的应用程序。为了获得对新噬菌体的免疫力，CRISPR相关蛋白(Cas1 和Cas2)整合噬菌体DNA片段(间隔区)在CRISPR基因座的“先导端”，靠近 转录起始点。但Cas1-2如何识别CRISPR的领导者端仍然知之甚少。 接下来，CRISPR基因座被转录并处理成“引导RNA”，加载到监控中 复合体(即CSM复合体)。当感应到病毒RNA时，CSM复合体产生环状寡核苷酸 调节CRISPR适应的信使和对噬菌体防御至关重要的核酸酶。但是生物学上的角色 其中许多免疫效应器仍未得到充分研究。CRISPR的生物信息学初步分析 Leaders(上游DNA)已经揭示了亚型和微生物特有的基序。这些保守的图案被发现 在相对于引线-CRISPR连接点的位置的紧密分布中，位置变量移位 DNA的螺旋转折，这将保持这些基序在DNA的同一“面”上的呈现。在目标1中，我 将确定生物化学捕获的CRISPR整合复合体的结构，以确定其作用 调节整合的先导基序和宿主蛋白。在目标2中，我将确定一种生化和结构 了解我预测将由CRISPR产生的核苷酸信使激活的免疫蛋白。 这一量身定制的应用程序将通过以下方式支持我过渡到独立的学术职位：i)结构 生物学培训，二)指导、交流和赠款撰写培训，三)鉴定新的CRISPR 适应调节基序和，iv)识别新的免疫效应器。我要正式的冷冻-EM 在蒙大拿州立大学(MSU)冷冻-EM设施提供的课程由共同顾问劳伦斯博士和 共同指导兰德博士，并在NIH的国家冷冻-EM访问和培训中心完成课程 训练。我将通过课程学习进一步发展我的指导、沟通和写作技能 由密歇根州立大学的教师卓越中心和ASBMB的UE5组件提供。我招募了一名 由六名在CRISPR生物学方面具有互补专业知识的科学家组成的指导和咨询委员会， 开发基于CRISPR的应用程序、冷冻-EM方法、病毒学和传染病- 当我追求我的研究和职业目标时，它与指导我相关。提议的项目为我提供了 低温EM结构生物学培训。它为我提供了基本的生物信息学和生化数据 将成为我的独立实验室对CRISPR生物学及其 申请。在那里，我的目标是成为下一代未被充分代表的少数族裔科学家的导师。