Mechanism, specificity, and design of CRISPR RNA-mediated gene regulation

CRISPR RNA介导的基因调控的机制、特异性和设计

基本信息

批准号：
9365125
负责人：
ILYA J FINKELSTEIN
金额：
$ 35.3万
依托单位：
UNIVERSITY OF TEXAS AT AUSTIN
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-15 至 2021-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9365125
关键词：
Adaptive Immune System Address Archaea Archaeal RNA Bacteria Bacterial RNA Binding Biochemical Bioinformatics Biology Biophysical Process Biophysics Biotechnology Cells Chromatin Cleaved cell Clustered Regularly Interspaced Short Palindromic Repeats Complex Crowding DNA DNA Binding DNA Sequence DNA-Binding Proteins Data Set Deoxyribonucleases Development Diffusion Dimensions Directed Molecular Evolution Electrostatics Elements Engineering Enzymes Eukaryotic Cell Family Future Gene Expression Gene Expression Regulation Gene Silencing Genes Genome Genome engineering Genomic Segment Genomics Goals Guide RNA Human Image Immune system Mediating Microscopy Modernization Molecular Mutation Nucleic Acids Nucleosomes Organism Play Process Proteins RNA Research Ribonucleases Role Scanning Site Specificity Structure System Techniques Testing Therapeutic Thymine Variant Virulence Virus adaptive immunity base clinical application design experimental study fluorescence imaging genetic manipulation genome editing imaging approach imaging platform improved insight interdisciplinary approach interest member nanoscale next generation novel nuclease pathogen precise genome editing predictive modeling programs protein profiling public health relevance single molecule tool

项目摘要

Project Summary Clustered regularly interspaced short palindromic repeats (CRISPRs) are a recently discovered RNA-based adaptive immune system that protects bacteria and archaea from foreign DNA. CRISPRs and the associated (Cas) proteins have been identified in 90% of archaea and 40% of bacteria, including many human pathogens. These immune systems also play a central role in controlling the horizontal transfer of virulence genes. The central step in CRISPR-Cas adaptive immunity is degradation of foreign DNA by a programmable RNA-guided nuclease. CRISPR-cas nucleases—enzymes that cleave a target DNA or RNA that is complementary to a guide CRISPR-RNA (crRNA)—have also been repurposed for precision gene regulation in many organisms. Despite the intense interest in these enzymes for both research and clinical applications, we still lack a complete understanding of their functions. Our long-term goal is to understand the mechanisms of CRISPR- mediated adaptive immunity. A related goal is to engineer improved CRISPR-associated enzymes for gene regulation and other biotechnological applications. The aims described in this proposal will mechanistically dissect a newly discovered family of RNA-guided nucleases. To achieve these aims, we pioneered high- throughput microscopy techniques that can image multiple enzymes and record their biochemical activities on tens of thousands of distinct DNA substrates. Using an interdisciplinary approach that integrates biophysics, bioinformatics, and micro-/nano-scale engineering, we will investigate how a CRISPR-associated RNA-guided nuclease recognizes and cleaves a target DNA. First, we will determine how the nuclease finds and cleaves a target DNA in the context of chromatin. Second, we will determine the biophysical mechanisms governing DNA binding and cleavage at off-target sites that resemble the on-target sequence. These off-target activities can cause unanticipated mutations that confound research experiments and limit therapeutic applications. Finally, we will engineer and profile novel high fidelity enzymes that reduce off-target activities and expand the genome engineering toolkit. CRISPR-mediated gene silencing and gene editing offers an exciting avenue for genetic manipulation of eukaryotic cells. We anticipate that our results will offer new insights in understanding the mechanisms of CRISPR-associated adaptive immunity and for using these enzymes for precision genome engineering in both scientific and future therapeutic settings.

项目摘要群集定期间隔短的短质体重复序列（CRISPRS）是最近发现的基于RNA的保护细菌和古细菌免受异物DNA的自适应免疫系统。 CRISPR和相关的（CAS）在90％的古细菌和40％的细菌中鉴定出蛋白质，包括许多人类病原体。这些免疫系统在控制病毒基因的水平转移方面也起着核心作用。 CRISPR-CAS适应性免疫学的中心步骤是通过可编程RNA引导的外国DNA降解核酸酶。 CRISPR-CAS核酶 - 清除靶DNA或RNA的酶指南CRISPR-RNA（CRRNA） - 在许多生物体中的精确基因调节也已被重新使用。尽管对这些酶对研究和临床应用都感兴趣，但我们仍然缺乏完全了解其功能。我们的长期目标是了解CRISPR的机制介导的适应性免疫。一个相关的目标是改善基因的CRISPR相关酶调节和其他生物技术应用。本提案中描述的目的将是机械机械的剖析了新发现的RNA引导的核所家族。为了实现这些目标，我们启动了高级可以对多种酶进行成像并记录其生化活动的吞吐量显微镜技术成千上万的不同DNA底物。使用整合生物物理学的跨学科方法，生物信息学和微型/纳米级工程，我们将研究如何与CRIS相关的RNA引入核酸酶识别并切割目标DNA。首先，我们将确定核酸酶如何找到和切割A 在染色质的背景下进行靶DNA。其次，我们将确定控制DNA的生物物理机制在类似于目标序列的脱靶位点上的结合和裂解。这些脱离目标的活动可以引起意外突变，使研究实验混淆并限制治疗应用。最后，我们将设计并介绍新颖的高保真酶，以减少脱靶活动并扩大基因组工程工具包。 CRISPR介导的基因沉默和基因编辑为通用的途径提供了令人兴奋的途径操纵真核细胞。我们预计我们的结果将为了解 CRISPR相关的适应性免疫学的机制，并将这些酶用于精确基因组在科学和未来的治疗环境中工程。