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.

项目概要 CRISPR-Cas 是原核适应性免疫系统，可保护细菌和古细菌免遭入侵 遗传元件，例如噬菌体和质粒。 CRISPR-Cas系统在过程中获得免疫记忆 通过将入侵者基因组的短片段整合到宿主的 CRISPR 基因座中来进行感染。这些 称为“间隔区”的片段随后被转录成 CRISPR RNA，加载到 Cas 核酸酶上并 引导它们识别并裂解感染性核酸。根据其基因组成，CRISPR- Cas系统分为六种类型（I-VI）。虽然间隔获取已在 I 型中进行了广泛研究 和II型系统一样，III型系统现在才刚刚开始探索。该应用程序的总体目标是 定义了控制间隔区获取的分子机制，该机制是流行但研究较少的 III-A 型 CRISPR-Cas 系统，并了解其在 CRISPR-Cas 防御和耐受过程中的影响。初步的 对表皮葡萄球菌 III-A 型系统的研究表明，该系统优先获得 两种独立模式的新垫片。第一种模式从一些但不是全部高度获得间隔物 转录基因，并跨越其整个转录区域。该提案的首要目的是阐明如何 收购机器将特定基因识别为优先收购的底物。这将是 通过解剖将间隔区整合酶复合物招募到特定基因的 DNA 序列来实现，发现 介导基因特异性间隔区获取的宿主因素，并测试该过程的生理相关性 在 CRISPR-Cas 免疫反应期间。 III-A型系统的第二种采集方式类似 之前研究的 I 型和 II 型系统，其中间隔区是从细菌的游离 dsDNA 末端获得的 染色体末端，其方式依赖于细胞的 DNA 修复机制。这样的自我定位 间隔区预计会诱导自身免疫并被负选择，但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 该位点的核酸酶，从而防止 CRISPR 自身免疫。该提案的第二个目标将定义 基因组环境允许自靶向间隔区被容忍，分析 CRISPR 的时间动态 游离 DNA 末端的 Cas 免疫，并探索 CRISPR 耐受性和所需的遗传成分 自靶向间隔物的积累。这项拟议的工作不仅会改变我们的概念理解 间隔子获取过程的一部分，但也可能导致基于 CRISPR 的分子技术发展 生物学和诊断学。为了实现这些目标，我组建了一支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。他们的指导以及持续的指导 Luciano Marraffini 教授的指导和洛克菲勒大学的科学环境，将使我能够表演 拟议的研究，以及培养写作、指导和沟通技巧，这将支持我的 成功过渡到独立职业。