Discovery of novel phage-bacterial interactions

发现新的噬菌体-细菌相互作用

• 批准号: 10282672
• 负责人: Nicole D. Marino
• 金额: $ 10万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-07 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10282672
• 关键词: Address; Affinity Chromatography; BCAR1 gene; Bacteria; Bacterial Proteins; Bacteriophages; Binding; Biological Assay; Biomedical Research; Candidate Disease Gene; Cells; Cessation of life; Cleaved cell; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complex; DNA Binding Domain; Evolution; Future; Genes; Genetic Engineering; Goals; Human; Immune; Immune system; Immunologic Tests; Individual; Infection; Lead; Lentivirus Vector; Libraries; Mass Spectrum Analysis; Measures; Mentors; Methodology; Methods; Molecular Biology; Nature; Oncogenes; Operon; Orthologous Gene; Outcome; Proteins; Regulator Genes; Reporter Genes; Research; Safety; System; Technology; Testing; Therapeutic Studies; Viral; Viral Vector; Virus; Work; Yeasts; base; cytotoxicity; endonuclease; gene discovery; gene therapy; genome editing; immune activation; immune system function; immunogenicity; improved; inhibitor/antagonist; insight; next generation sequencing; novel; off-target mutation; post-doctoral training; programs; protein protein interaction; reconstitution; restriction enzyme; side effect; tool; transcription factor; vector; yeast two hybrid system

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.

项目概要 细菌与其病毒（噬菌体）之间的相互作用是自然界中最普遍存在的相互作用，并且 产生了基因工程的变革性工具，例如限制性内切酶和 CRISPR-Cas。 最近在许多细菌物种中发现了三打新的细菌免疫系统。 其中一些免疫系统（例如 CRISPR）以噬菌体为目标进行切割，而其他免疫系统则感知噬菌体 感染并诱导细菌死亡以阻止噬菌体传播，反过来，噬菌体表达“抗免疫”蛋白质。 解除这些细菌的防御，包括抑制 Cas 效应器功能的“抗 CRISPR”(Acr) 蛋白。 对于大多数这些免疫，尚未发现噬菌体衍生的相互作用物（抑制剂或激活剂） 然而，系统。 该提案的长期目标是鉴定与噬菌体蛋白相互作用或触发其激活的噬菌体蛋白。 这些免疫系统将使用酵母两种杂交筛选来识别并验证。 使用亲和纯化-质谱分析细菌。在双杂交筛选中，Gal4。 转录因子将被分成激活结构域和 DNA 结合结构域并融合到每个噬菌体 分别是“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能够表达报告基因，从而赋予 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因子的免疫蛋白将加速这一过程。 噬菌体与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，噬菌体用于灭活细菌中 CRISPR-Cas 系统的策略将应用于基因 在人体细胞中进行治疗，以减少细胞毒性和脱靶效应，从而使 Cas9 持续失活。 细菌中的 Cas12a 通常会阻断靶向和表达，这可能是长期 Cas 的最佳选择 使用病毒载体传递的 Cas9 进行的哺乳动物基因编辑通常会导致脱靶。 为了减轻这些脱靶效应， 灭活 CRISPR-Cas 复合物并减少其表达的策略（在靶向编辑完成后） 这项工作将在世界一流的加州大学旧金山分校进行。 它也将为我提供设施和高度智力和协作的研究社区。 我需要完成博士后培训的蛋白质-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项目。