Generation of immunological memory by CRISPR-Cas systems

项目摘要 CRISPR-CAS基因座由短DNA重复组成，由相等的短序列(称为间隔区)隔开 匹配原核生物病毒(噬菌体)和质粒的基因组，并提供基于序列的免疫 对抗这些元素。免疫是由小的反义CRISPR RNA分子(CrRNAs)介导的，这些分子 从间隔区转录，并将CRISPR相关(CAS)核酸酶引导到入侵的核酸中 乳沟和破坏。在我的博士后研究期间，我开创了CRISPR-CAS系统的研究，以 为这种细菌免疫途径奠定基础。利用遗传学，我确定CRISPR-CAS 系统以序列特异性的方式靶向DNA分子，这项研究是理解 CRISPR免疫分子水平的机制。这一发现预言了RNA的存在-- 可编程CaS核酸酶及其在基因组编辑中的当前应用。 在质粒或噬菌体感染时，CRISPR-Cas系统结合了与 入侵者的基因组。这一过程记录了感染的记忆，随后用于生成 CrRNA为CaS核酸酶提供指导。虽然识别和识别背后的分子机制 被Cas核酸酶切割的靶序列是很好理解的，宿主如何获得新的 间隔区；即CRISPR-Cas免疫反应的免疫阶段，仍然是一个谜。在这份提案中，我 计划研究原核宿主如何从入侵者那里获得新的间隔区序列，使用组合 分子遗传学和下一代测序方法。基本问题，如(I)如何 自身免疫，或从宿主染色体上获得间隔区，被阻止；(Ii) 免疫过程与病毒感染周期的比较；(3)其他哪些细胞途径，如果有的话，帮助 CRISPR免疫；(Iv)如何从入侵者的基因组中提取新的间隔区序列；以及(V)如何 免疫过程影响宿主人口的演变；尚未得到回答。建议数 研究将极大地促进我们对CRISPR-CAS分子机制的理解 免疫以及这些基因座对原核生物生态和进化的影响 把他们藏起来。此外，我们的实验将要求或允许我们设计CRISPR-CAS系统 进行高频间隔器采集。这种系统将促进技术的发展。 具有需要将特定细胞事件记录到特定基因组位置的应用 追踪长期细胞历史的研究人员。因此，拟议的研究可以提供新的开发基础。 CRISPR免疫接种用于革命性的生物技术和/或治疗目的。

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.