Molecular mechanisms of memory formation and tolerance in CRISPR-Cas systems

CRISPR-Cas系统中记忆形成和耐受的分子机制

• 批准号: 10570544
• 负责人: Naama Aviram
• 金额: $ 12.5万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10570544
• 关键词: Adaptive Immune System; Affect; Archaea; Autoimmunity; Bacteria; Bacterial Genome; Bacteriophages; Biochemical; Biochemistry; Bioinformatics; Biology; Biophysics; CRISPR/Cas technology; Cells; Characteristics; Classification; Clustered Regularly Interspaced Short Palindromic Repeats; Communication; Complex; DNA; DNA Repair; DNA Sequence; DNA-Directed RNA Polymerase; Data; Development; Elements; Engineering; Environment; Genes; Genetic; Genetic Transcription; Genome; Genomics; Goals; Health; Human; Immune response; Immune system; Immunity; Immunologic Memory; Impairment; Infection; Integrase; Integration Host Factors; Invaded; Laboratories; Link; Location; Mediating; Memory; Mentorship; Mobile Genetic Elements; Molecular; Molecular Biology; Nucleic Acids; Outcome; Pathogenicity; Physiological; Plasmids; Population; Process; RNA; Research; Research Design; Ribosomal RNA; Role; Site; Staphylococcus epidermidis; System; Testing; Transcript; Transfer RNA; Universities; Work; Writing; biological systems; career; cold shock protein; cost; ds-DNA; experimental study; fitness; genome editing; genome wide screen; genotoxicity; improved; insight; memory acquisition; molecular diagnostics; nuclease; prevent; promoter; recruit; recurrent infection; skills; technology development; tool

Project Summary CRISPR-Cas are prokaryotic adaptive immune systems that protect bacteria and archaea from invading mobile genetic elements, such as phages and plasmids. CRISPR-Cas systems acquire immunological memories during infection by integrating short fragments from the invader’s genome into the CRISPR locus of the host. These fragments, called “spacers”, are later transcribed into CRISPR RNAs that are loaded on Cas nucleases and guide them to recognize and cleave infecting nucleic acids. Depending on their genetic composition, CRISPR- Cas systems are classified into six types (I-VI). While spacer acquisition has been extensively studied in type I and II systems, type III systems are just now starting to be explored. The overall goal of this application is to define the molecular mechanisms that govern spacer acquisition by the prevalent, yet less studied, type III-A CRISPR-Cas system, and understand its implications during CRISPR-Cas defense and tolerance. Preliminary work on the type III-A system of Staphylococcus epidermidis revealed that this system preferentially acquires new spacers by two independent modes. The first mode acquires spacers from some, but not all, highly transcribed genes, and spans their entire transcribed region. The first aim of this proposal is to elucidate how the acquisition machinery recognizes specific genes as substrates for preferential acquisition. This will be achieved by dissecting the DNA sequences that recruit the spacer-integrase complex to specific genes, finding host factors that mediate gene-specific spacer acquisition, and test for the physiological relevance of this process during the CRISPR-Cas immune response. The second mode of acquisition by the type III-A system is similar to the previously studied type I and II systems, where spacers are acquired from free dsDNA ends at the bacterial chromosomal terminus, in a manner that is dependent on the cell’s DNA-repair machinery. Such self-targeting spacers are expected to induce autoimmunity and be negatively selected, however we found them to be stably fixed in the bacterial population, suggesting the existence of unknown mechanisms that inhibit targeting by Cas nucleases at this site, thus preventing CRISPR autoimmunity. The second aim of this proposal will define the genomic context that allows self-targeting spacers to be tolerated, analyze the temporal dynamics of CRISPR- Cas immunity at free DNA ends, and explore the genetic components needed for CRISPR-tolerance and accumulation of self-targeting spacers. This proposed work will not only transform our conceptual understanding of the spacer acquisition process, but also could lead to CRISPR-based technological developments in molecular biology and diagnostics. To achieve these goals, I have assembled a team of experts in the fields of transcription, DNA repair, bioinformatics, biochemistry and biophysics. Their guidance, along with the continued mentorship of Prof. Luciano Marraffini and the scientific environment of the Rockefeller University, will allow me to perform the proposed research, as well as to develop writing, mentorship and communication skills, that will support my successful transition to an independent career.

项目摘要 CRISPR-Cas是原核适应性免疫系统，可保护细菌和古细菌免受移动的入侵 遗传因素，如DNA和质粒。CRISPR-Cas系统在免疫过程中获得免疫记忆 通过将来自入侵者基因组的短片段整合到宿主的CRISPR基因座中来感染。这些 称为“间隔区”的片段随后被转录成CRISPR RNA，CRISPR RNA装载在Cas核酸酶上， 引导它们识别并切割感染性核酸。根据它们的基因组成，CRISPR- Cas系统分为六种类型（I-VI）。虽然间隔区的获取在I型糖尿病中已经被广泛研究， 和II型系统，III型系统才刚刚开始探索。此应用程序的总体目标是 定义了流行但研究较少的III-A型控制间隔区获取的分子机制 CRISPR-Cas系统，并了解其在CRISPR-Cas防御和耐受过程中的意义。初步 对表皮葡萄球菌的III-A型系统的研究表明，该系统优先获得 两种独立模式的新型间隔器。第一种模式从一些但不是所有高度依赖的细胞中获得间隔物。 转录基因，并跨越其整个转录区域。本建议的第一个目的是阐明如何 获取机制识别特定基因作为优先获取的底物。这将是 通过解剖将间隔子整合酶复合物募集到特定基因的DNA序列， 介导基因特异性间隔区获得的宿主因子，并测试该过程的生理相关性 在CRISPR-Cas免疫反应期间。III-A类系统的第二种获取模式与此类似 先前研究的I型和II型系统，其中间隔区是从细菌DNA末端的游离dsDNA末端获得的。 染色体末端，以依赖于细胞的DNA修复机制的方式。这种自我定位 预期间隔区诱导自身免疫并被阴性选择，然而，我们发现它们是稳定的。 固定在细菌群体中，表明存在抑制Cas靶向的未知机制 因此，CRISPR可以在该位点上产生核酸酶，从而防止CRISPR自身免疫。本提案的第二个目标是确定 允许自我靶向间隔区被容忍的基因组背景，分析CRISPR的时间动态- Cas游离DNA末端的免疫力，并探索CRISPR耐受所需的遗传成分， 自靶向间隔区的积累。这项拟议中的工作不仅将改变我们的概念理解， 间隔区获取过程，但也可能导致基于CRISPR的分子生物学技术的发展。 生物学和诊断学为了实现这些目标，我组建了一个转录领域的专家团队， DNA修复、生物信息学、生物化学和生物物理学。他们的指导，沿着着持续的指导 卢西亚诺·马拉菲尼教授和洛克菲勒大学的科学环境，将允许我表演 拟议的研究，以及发展写作，指导和沟通技巧，这将支持我的 成功转型为独立职业。