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

摘要 结核分枝杆菌(Mtb)基因表达的调控仍然是一个挑战。特别是 同时调控多个基因是困难的，也是非常耗时的。CRISPR-CAS 系统是一个RNA引导的细菌适应性免疫系统，具有改造基因编辑的潜力，以及 CRISPR干扰(CRISPRi)，是一种控制多种生物基因表达的新工具。CRISPR 系统本质上是多基因调控系统，基于它们表达和处理CRISPR的能力 排列成几十个RNA，每个RNA可以针对不同的基因。这种独特的多基因打靶功能 尚未被利用，因为并不是所有必需的前体RNA加工组件都是已知的 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 当所有成分需要异源表达时，CRISPR系统的正交使用。内生性 相比之下，CRISPR系统提供处理和干扰的所有必要组件，并 经过优化调整，以在其特定的宿主中发挥作用。在这里，我们将重新定位内源性结核分枝杆菌III型 CRISPR系统，用于任何菌株的基因调控和多基因调控。

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.