Discovery of novel phage-bacterial interactions

项目摘要 细菌和它们的病毒之间的相互作用（细菌）是自然界中最普遍存在的， 产生了基因工程的变革性工具，如限制性内切酶和CRISPR-Cas。超过 最近在许多细菌物种中发现了36种新的细菌免疫系统。 这些免疫系统中的一些，如CRISPR，靶向噬菌体进行切割，而另一些则感测噬菌体。 感染并诱导细菌死亡以阻止噬菌体传播。反过来，EMPs表达“抗免疫”蛋白质， 解除这些细菌防御，包括抑制Cas效应子功能的“抗CRISPR”（Acr）蛋白。 噬菌体衍生的相互作用物（抑制剂或激活剂）尚未被发现用于大多数这些免疫反应。 然而，系统。 这项提议的长期目标是鉴定与噬菌体蛋白相互作用或触发噬菌体蛋白激活的噬菌体蛋白。 这些免疫系统。这些相互作用将使用酵母双杂交筛选进行鉴定和验证 在细菌中使用亲和纯化-质谱分析。在双杂交屏幕中，Gal 4 转录因子将被分成激活结构域和DNA结合结构域，并与每个噬菌体融合 “猎物”蛋白和细菌免疫“诱饵”蛋白。猎物蛋白和诱饵之间的相互作用 蛋白质应该重建完整的转录因子，并使报告基因的表达，赋予 选择性媒体的生存。将筛选合理选择的噬菌体蛋白与CRISPR的相互作用。 Cas蛋白以及缺乏已知相互作用物的免疫蛋白。这个多功能平台将加速 噬菌体-细菌相互作用的发现，长期以来改变了分子生物学和基因治疗。 与此同时，噬菌体用于在细菌中抑制CRISPR-Cas系统的策略将应用于基因工程。 在人类细胞中进行治疗以减少细胞毒性和脱靶效应。组成性地抑制Cas9的噬菌体 而细菌中的Cas 12 a通常会阻断靶向和表达，这可能是长期Cas的最佳选择。 失活用在病毒载体上递送的Cas9进行的哺乳动物基因编辑通常会导致脱靶 与长期Cas9表达相关的突变和细胞毒性。为了减轻这些脱靶效应， 抑制CRISPR-Cas复合物并减少其表达的策略（在靶向编辑后， 将进行合并和比较。这项工作将在加州大学旧金山分校进行，该大学拥有世界级的 设施和高度智力和协作的研究社区。它还将为我提供 蛋白质相互作用筛选和基因编辑方面的专业知识，我需要完成我的博士后培训 目标，并开创了细菌噬菌体相互作用的独立研究计划。

PROJECT SUMMARY Interactions between bacteria and their viruses (phages) are among the most ubiquitous in nature and have yielded transformative tools for genetic engineering, such as restriction enzymes and CRISPR-Cas. More than three dozen new bacterial immune systems have recently been discovered across many bacterial species. Some of these immune systems, such as CRISPR, target phage for cleavage, while others sense phage infection and induce bacterial death to halt phage spread. In turn, phages express “anti-immune” proteins to disarm these bacterial defenses, including “anti-CRISPR” (Acr) proteins that inhibit Cas effector functions. Phage-derived interactors (either inhibitors or activators) have not yet been found for most of these immune systems, however. The long-term objective of this proposal is to identify phage proteins that interact with or trigger activation of these immune systems. These interactions will be identified using yeast two hybrid screens and validated using affinity purification-mass spectrometry analysis in bacteria. In the two-hybrid screen, the Gal4 transcription factor will be split into an activation domain and DNA-binding domain and fused to each phage “prey” protein and bacterial immune “bait” protein, respectively. Interaction between the prey protein and bait protein should reconstitute the full transcription factor and enable expression of a reporter gene that confers survival on selective media. Rationally selected phage proteins will be screened for interactions with CRISPR- Cas proteins as well as immune proteins that lack known interactors. This versatile platform will accelerate the discovery of phage-bacterial interactions, which have long transformed molecular biology and gene therapy. In parallel, the strategies that phage use to inactivate CRISPR-Cas systems in bacteria will be applied to gene therapy in human cells to reduce cytotoxicity and off-target effects. Phages that constitutively inactivate Cas9 and Cas12a in bacteria often block both targeting and expression, which is likely optimal for long-term Cas inactivation. Mammalian gene editing performed with Cas9 delivered on viral vectors often causes off-target mutations and cytotoxicity associated with long-term Cas9 expression. To mitigate these off-target effects, strategies to inactivate CRISPR-Cas complexes and reduce their expression (after on-target editing has occurred) will be combined and compared. This work will be performed at UCSF, which hosts world-class facilities and a highly intellectual and collaborative research community. It will also provide me with the expertise in protein-protein interaction screens and gene editing that I need to fulfill my postdoctoral training goals and pioneer an independent research program in bacterial-phage interactions.