Multigene knockdown in Mycobacterium tuberculosis by repurposing the endogenous type III CRISPR system

通过重新利用内源 III 型 CRISPR 系统实现结核分枝杆菌多基因敲除

• 批准号: 9308467
• 负责人: Christoph Grundner
• 金额: $ 9.8万
• 依托单位: SEATTLE BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9308467
• 关键词: Adaptive Immune System; Anti-HIV Agents; CRISPR interference; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complementary RNA; Enzymes; Gene Chips; Gene Expression; Gene Expression Regulation; Gene Family; Gene Targeting; Genes; Genetic; Genetic Transcription; Guide RNA; Mycobacterium tuberculosis; Organism; Pathogenesis; Precursor RNA; Process; Proteins; Public Health; RNA; RNA Precursors; RNA Processing; Regulation; Research; Self-Direction; Series; Specific qualifier value; System; Testing; Time; Toxin; Transcript; Transcriptional Regulation; Tuberculosis; base; design; drug development; endonuclease; knock-down; member; tool; transcriptome sequencing

ABSTRACT The manipulation of gene expression in Mycobacterium tuberculosis (Mtb) remains a challenge. In particular the regulation of multiple genes simultaneously is difficult and prohibitively time consuming. The CRISPR-Cas system is an RNA-guided bacterial adaptive immune system with transformative potential for gene editing, and CRISPR interference (CRISPRi), is a new tool to control gene expression in many organisms. CRISPR systems are inherently multi-gene regulatory systems, based on their ability to express and process a CRISPR array into many dozen RNAs that can each target a different gene. This unique multi-gene targeting function has not yet been exploited because not all required precursor RNA processing components are known for most systems, precluding their heterologous expression. However, these limitations apply only to the orthogonal use of CRISPR systems when heterologous expression of all components is required. Endogenous CRISPR systems, in contrast, provide all necessary components for processing and interference and are optimally tuned to function in their particular host. Here, we will repurpose the endogenous Mtb type III CRISPR system for the regulation of genes in any strain and for multi-gene regulation.

摘要 结核分枝杆菌（Mycobacterium tuberculosis，Mtb）的基因表达调控仍是一个挑战。特别是 同时调节多个基因是困难的，并且极其耗时。该CRISPR-Cas 系统是RNA引导的细菌适应性免疫系统，具有基因编辑的变革潜力， CRISPR干扰（CRISPRi）是一种控制许多生物体中基因表达的新工具。CRISPR CRISPR系统本质上是多基因调控系统，基于其表达和加工CRISPR的能力， 排列成几十个RNA，每个RNA可以靶向不同的基因。这种独特的多基因靶向功能 还没有被利用，因为不是所有需要的前体RNA加工组分都是已知的， 大多数系统，排除了它们的异源表达。但是，这些限制仅适用于 当需要所有组分的异源表达时，可以正交使用CRISPR系统。内源 相反，CRISPR系统提供了处理和干扰的所有必要组件， 最佳地调整以在其特定宿主中发挥作用。在这里，我们将重新利用内源性Mtb III型 CRISPR系统用于任何菌株中的基因调控和多基因调控。