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

项目摘要 CRISPR-CA是保护细菌和古菌免受入侵的原核适应性免疫系统 遗传因素，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基因座上而感染。这些 片段，称为“间隔区”，后来被转录成CRISPR RNA，装载在CAS核酸酶上，并 引导他们识别和裂解感染性核酸。根据它们的遗传组成，CRISPR- CAS系统分为六种类型(I-VI)。虽然间隔区获得已经在类型I中得到了广泛的研究 和第二类系统，第三类系统才刚刚开始探索。此应用程序的总体目标是 确定控制流行的但研究较少的III-A型间隔区获得的分子机制 CRISPR-CAS系统，并了解其在CRISPR-CAS防御和容忍过程中的含义。初步 对Ⅲ-A型表皮葡萄球菌系统的研究表明，该系统优先获得 两种独立模式的新间隔器。第一种模式从一些但不是全部高度获得间隔体 转录基因，并跨越其整个转录区域。这项提议的第一个目的是阐明 获取机制识别特定的基因作为优先获取的底物。这将是 通过解剖将间隔区整合酶复合体招募到特定基因的DNA序列，发现 介导基因特异性间隔区获得的宿主因素，并测试这一过程的生理学相关性 在CRISPR-Cas免疫反应过程中。III型-A型系统的第二种采集模式类似 对于先前研究的I型和II型系统，其中间隔物从细菌的游离dsdna末端获得 染色体末端，以依赖于细胞DNA修复机制的方式。这样的自我瞄准 间隔区被认为是诱导自身免疫和负选择的，但我们发现它们是稳定的 固定在细菌种群中，表明存在未知的机制，抑制了CA的靶向 核酸酶，从而阻止CRISPR自身免疫。该提案的第二个目标将定义 允许耐受自定位间隔区的基因组环境，分析CRISPR- 在游离DNA末端的CAS免疫，并探索CRISPR耐受和 自我定位间隔体的积累。这项拟议的工作不仅将改变我们对 间隔区获取过程的研究，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，以及持续的指导 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、指导和沟通技能，这将支持我的 成功过渡到独立的职业生涯。

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.