Generation of immunological memory by CRISPR-Cas systems

项目摘要 CRISPR-Cas基因座由短DNA重复序列组成，这些短DNA重复序列被等短序列（称为间隔子）隔开， 匹配原核病毒和质粒的基因组，并赋予基于序列的免疫力 反对这些元素。免疫是由小的反义CRISPR RNA分子（crRNA）介导的， 从间隔区转录并引导CRISPR相关（Cas）核酸酶至入侵核酸， 分裂和毁灭在我的博士后研究期间，我开创了CRISPR-Cas系统的研究， 为这种细菌免疫途径奠定了基础。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，这项研究是了解 CRISPR免疫在分子水平上的作用机制。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。 在质粒或噬菌体感染后，CRISPR-Cas系统掺入与质粒或噬菌体感染相匹配的新的间隔子序列。 入侵者的基因组该过程记录感染的记忆，随后用于生成 Cas核酸酶的crRNA向导。虽然识别和识别的分子机制 Cas核酸酶对靶序列的切割是很好理解的，宿主如何能够获得新的 CRISPR-Cas免疫应答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， 分子遗传学和下一代测序方法的重要性。基本问题，如（i）如何 自身免疫性，或从宿主染色体获得间隔区，被阻止;（ii） 与病毒感染周期相比，免疫过程;（iii）其他细胞途径（如果有的话） CRISPR免疫;（iv）如何从入侵者的基因组中采样新的间隔序列;以及（v）如何 免疫过程影响宿主种群的进化;尚未得到回答。拟议 这些研究将大大推进我们对CRISPR-Cas基础分子机制的理解， 免疫以及这些基因座对原核生物生态学和进化的影响， 庇护他们此外，我们的实验将要求或允许我们设计CRISPR-Cas系统， 以高频率执行间隔物采集。这种系统将促进技术的发展 其应用需要将特定细胞事件记录到特定基因组位点中 研究人员追踪了细胞的长期历史。因此，拟议的研究可以提供新的地面利用 CRISPR免疫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.