Discovery of novel phage-bacterial interactions

项目总结 细菌和它们的病毒(噬菌体)之间的相互作用是自然界中最普遍的相互作用之一，并具有 产生了遗传工程的变革性工具，如限制性内切酶和CRISPR-CAS。多过 最近在许多细菌物种中发现了30多个新的细菌免疫系统。 其中一些免疫系统，如CRISPR，以噬菌体为切割目标，而另一些则感知噬菌体 感染并诱导细菌死亡，以阻止噬菌体的传播。反过来，噬菌体表达“抗免疫”蛋白以 解除这些细菌防御，包括抑制Cas效应器功能的“抗CRISPR”(ACR)蛋白。 噬菌体衍生的相互作用因子(抑制物或激活剂)对大多数免疫作用尚未被发现。 然而，系统。 这项提议的长期目标是识别与之相互作用或触发激活的噬菌体蛋白。 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 利用亲和纯化-质谱法对细菌进行分析。在双混合屏幕上，Gal4 转录因子将被分成活化域和DNA结合域，并与每个噬菌体融合 “猎物”蛋白和细菌免疫“诱饵”蛋白分别。猎物蛋白与诱饵的相互作用 蛋白质应该重新构成完整的转录因子，并能够表达一个报告基因 在有选择的媒体上生存。合理选择的噬菌体蛋白将被筛选与CRISPR- CAS蛋白以及缺乏已知相互作用的免疫蛋白。这一多功能平台将加速 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。 同时，噬菌体用来灭活细菌中CRISPR-Cas系统的策略将应用于基因 在人类细胞中进行治疗，以减少细胞毒性和靶外效应。结构性灭活Cas9的噬菌体 细菌中的Cas12a通常同时阻止靶向和表达，这可能是长期CA的最佳选择 失活。用病毒载体上的Cas9进行哺乳动物基因编辑经常导致脱靶 与Cas9长期表达相关的突变和细胞毒性。为了减轻这些偏离目标的影响， 灭活CRISPR-CAS复合体并减少其表达的策略(在靶标编辑后 发生的)将被组合和比较。这项工作将在加州大学旧金山分校进行，它主办了世界级的 设施和高度智能化和协作性的研究社区。它还将为我提供 完成博士后培训所需的蛋白质-蛋白质相互作用筛选和基因编辑方面的专业知识 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.