Generation of immunological memory by CRISPR-Cas systems

通过 CRISPR-Cas 系统生成免疫记忆

• 批准号: 9750114
• 负责人: Luciano A Marraffini
• 金额: $ 118.65万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-30 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9750114
• 关键词: Affect; Autoimmunity; Bacteria; Bacteriophages; Base Sequence; Biotechnology; Cells; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; Ecology; Elements; Engineering; Event; Evolution; Foundations; Frequencies; Generations; Genetic; Genetic Diseases; Genome; Genomics; Health; Human; Human Genetics; Immune response; Immune system; Immunity; Immunization; Immunologic Memory; Infection; Invaded; Knowledge; Mediating; Memory; Molecular; Molecular Genetics; Mutation; Nucleic Acid Cleavage; Organism; Pathway interactions; Phase; Plasmids; Population; Postdoctoral Fellow; Process; Prokaryotic Cells; RNA; Recording of previous events; Records; Research Personnel; Sampling; System; Therapeutic; Viral; Virus; base; experimental study; genome editing; next generation sequencing; nuclease; prevent; programs; technology development

Project Summary CRISPR-Cas loci consist of short DNA repeats separated by equally short sequences (known as spacers) that match the genomes of prokaryotic viruses (phages) and plasmids and confer sequence-based immunity against these elements. Immunity is mediated by small, antisense CRISPR RNA molecules (crRNAs) that are transcribed from spacers and guide CRISPR-associated (Cas) nucleases to the invading nucleic acid for cleavage and destruction. During my post-doctoral studies I pioneered the study of CRISPR-Cas systems to establish the foundations of this bacterial immunity pathway. Using genetics, I determined that CRISPR-Cas systems target DNA molecules in a sequence-specific manner, a study that was key to understand the mechanisms of CRISPR immunity at the molecular level. This finding predicted the existence of RNA- programmable Cas nucleases and their current applications to genome editing. Upon plasmid or phage infection, CRISPR-Cas system incorporate new spacer sequences that match the genome of the invader. This process records a memory of the infection that is subsequently used to generate the crRNA guides for the Cas nucleases. While the molecular mechanisms behind the recognition and cleavage of target sequences by the Cas nucleases are well understood, how the host can acquire new spacers; i.e. the immunization phase of the CRISPR-Cas immune response, is still a mystery. In this proposal I plan to study how the prokaryotic host acquires new spacer sequences from its invaders, using a combination of molecular genetics and next-generation sequencing approaches. Fundamental questions such as (i) how autoimmunity, or the acquisition of spacers from the host chromosome, is prevented; (ii) how fast is the immunization process compared to the viral infectious cycle; (iii) which other cellular pathways, if any, assist CRISPR immunization; (iv) how new spacer sequences are sampled from the invader's genome; and (v) how the immunization process affects the evolution of the host population; are not yet answered. The proposed studies will substantially advance our understanding of the molecular mechanisms underlying CRISPR-Cas immunization and the impact that these loci have on the ecology and evolution of prokaryotes organisms that harbor them. In addition, our experiments will require or allow us to engineer CRISPR-Cas systems that perform spacer acquisition with high frequency. Such systems will facilitate the development of technologies with applications that require the recording of specific cellular events into a specific genomic locus to enable researchers following long cellular histories. Thus the proposed studies could provide new ground to exploit CRISPR immunization for revolutionary biotechnological and/or therapeutic purposes.

项目概要 CRISPR-Cas 位点由由同样短的序列（称为间隔区）隔开的短 DNA 重复序列组成， 匹配原核病毒（噬菌体）和质粒的基因组并赋予基于序列的免疫力 反对这些要素。免疫是由小的反义 CRISPR RNA 分子 (crRNA) 介导的，这些分子 从间隔区转录并引导 CRISPR 相关 (Cas) 核酸酶到达入侵核酸 分裂和破坏。在我的博士后研究期间，我开创了 CRISPR-Cas 系统的研究 建立这种细菌免疫途径的基础。利用遗传学，我确定 CRISPR-Cas 系统以特定序列的方式靶向 DNA 分子，这项研究对于理解 DNA 分子的关键 分子水平上的 CRISPR 免疫机制。这一发现预测了RNA的存在 可编程 Cas 核酸酶及其当前在基因组编辑中的应用。 质粒或噬菌体感染后，CRISPR-Cas 系统会结合新的间隔序列来匹配 入侵者的基因组。该过程记录了感染的记忆，随后用于生成 Cas 核酸酶的 crRNA 指导。虽然识别和识别背后的分子机制 Cas核酸酶对靶序列的切割已被充分理解，宿主如何获得新的 垫片；即 CRISPR-Cas 免疫反应的免疫阶段仍然是一个谜。在这个提案中我 计划研究原核宿主如何使用组合从其入侵者那里获取新的间隔序列 分子遗传学和下一代测序方法。基本问题，例如 (i) 如何 防止自身免疫或从宿主染色体获得间隔区； (ii) 速度有多快 免疫过程与病毒感染周期的比较； (iii) 哪些其他细胞途径（如果有的话）可以提供帮助 CRISPR免疫； (iv) 如何从入侵者的基因组中取样新的间隔序列； (v) 如何 免疫过程影响宿主种群的进化；尚未得到答复。拟议的 研究将极大地增进我们对 CRISPR-Cas 分子机制的理解 免疫以及这些位点对原核生物的生态和进化的影响 庇护他们。此外，我们的实验将需要或允许我们设计 CRISPR-Cas 系统， 以高频执行间隔获取。这样的系统将促进技术的发展 需要将特定细胞事件记录到特定基因组位点的应用程序 研究人员追踪长期的细胞历史。因此，拟议的研究可以提供新的利用基础 用于革命性生物技术和/或治疗目的的 CRISPR 免疫。