Mechanism of Co-Transcriptional DNA and RNA Targeting by Type III CRISPR-Cas Systems

III 型 CRISPR-Cas 系统共转录 DNA 和 RNA 靶向机制

• 批准号: 9259287
• 负责人: Tina Y Liu
• 金额: $ 5.71万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9259287
• 关键词: Adaptive Immune System; Affect; BCAR1 gene; Bacteria; Bacterial RNA; Bacteriophages; Biochemistry; Biological Assay; Cell physiology; Cells; Cleaved cell; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Complex; Cryoelectron Microscopy; DNA; DNA Binding; DNA-Directed RNA Polymerase; Deoxyribonucleases; Development; Diabetes Mellitus; Disease; Electron Transport Complex III; Escherichia coli; Gene Expression; Genetic; Genetic Transcription; Genome; Guide RNA; Heart Diseases; Human; Imagery; In Vitro; Infection; Lead; Malignant Neoplasms; Messenger RNA; Mobile Genetic Elements; Modeling; Nucleic Acid Cleavage; Nucleic Acids; Organism; Physiological; Prokaryotic Cells; RNA; RNA Binding; Recruitment Activity; Reporting; Resolution; Role; Structure; System; Testing; Thermus thermophilus; Time; Tissues; Transcript; Work; adaptive immunity; disorder risk; genome analysis; in vitro activity; in vivo; insight; nucleic acid binding protein; particle; programs; protein complex; reconstitution; tool

PROJECT SUMMARY Prokaryotes use an adaptive immune system called CRISPR-Cas to defend against phages and other mobile genetic elements. These systems acquire short pieces of DNA from their invaders as a genetic record of prior infections. They use these to produce CRISPR RNAs that are assembled with one or more Cas proteins into RNA-guided interference complexes that find and cleave a complementary target in foreign DNA or RNA. While most types of CRISPR-Cas systems target only one type of nucleic acid, Type III CRISPR systems have been reported to target both DNA and RNA. The identity of the true target nucleic acid of Type III systems thus remains unknown. A bacterial Type III-A system was reported to target transcriptionally active loci in vivo, leading to the hypothesis that the transcription bubble, at which DNA and RNA are both present, is the physiological target. Target recognition by the interference complex thus may involve simultaneous recognition of both DNA and RNA. In order to test this model, we propose to 1) trap a Type III-A (Csm) complex bound to a transcriptionally active target and use cryo-electron microscopy to determine the structure of the complex and 2) use protein-nucleic acid binding and nucleic acid cleavage assays to determine if RNA binding recruits the Type III-A (Csm) complex to the transcription bubble. This would lay the groundwork for repurposing these systems to detect transcriptionally active loci, and regulate gene expression in heterologous organisms.

项目摘要 原核生物使用一种名为CRISPR-Cas的适应性免疫系统来防御细菌和其他移动的 遗传因素这些系统从它们的入侵者那里获得短的DNA片段，作为先前入侵者的遗传记录。 感染.他们使用这些来产生CRISPR RNA，这些RNA与一种或多种Cas蛋白组装成 RNA引导的干扰复合物，发现并切割外源DNA或RNA中的互补靶标。 虽然大多数类型的CRISPR-Cas系统仅靶向一种类型的核酸，但III型CRISPR系统具有靶向一种类型的核酸的能力。 据报道，它可以靶向DNA和RNA。因此，III型系统的真正靶核酸的身份 仍然未知。据报道，细菌III-A型系统靶向体内转录活性位点， 这导致了这样一种假设，即DNA和RNA都存在的转录泡是转录泡。 生理目标因此，干扰复合物的目标识别可能涉及同时识别 DNA和RNA。为了测试这个模型，我们建议：1）捕获一个III-A型（CSM）复合物， 转录活性靶点，并使用冷冻电子显微镜来确定复合物的结构， 2)使用蛋白质-核酸结合和核酸切割测定来确定RNA结合是否募集了 III-A型（CSM）复合物的转录泡。这将为重新利用这些 检测转录活性位点和调节异源生物体中基因表达的系统。