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使用I-E型CRISPR系统在多倍体,耐辐射细菌Deinococcus radiodurans中的有效基因沉默

Chitra S Misra¹, Neha Pandey^1,2, Deepti Appukuttan³

¹Applied Genomics Section, Bio-Science Group, Bhabha Atomic Research Centre , Mumbai, Maharashtra, India.

Microbiology spectrum

|September 6, 2023

概括

我们开发了一种CRISPR-Cas系统,用于对抗辐射的细菌Deinococcus radiodurans的基因敲除. 这种工具有效地减少了基因表达,帮助研究辐射反应和工业应用.

科学领域:

微生物学微生物学
分子生物学分子生物学
生物技术是生物技术.

背景情况:

迪诺科克 (Deinococcus radiodurans) 是一种具有工业潜力的高度耐辐射的细菌.
由于其多倍体基因组,有限的分子工具阻碍了D. radiodurans的基因操纵.
集群定期间隔的短平行体重复 (CRISPR) -Cas系统提供了多功能基因操纵能力.

研究的目的:

开发和演示一种CRISPR-Cas型I-E级联系统,用于对D. radiodurans的基因敲除.
评估该系统在沉默特定基因和调节元件方面的有效性.
建立一种多功能工具,用于研究辐射反应,并使D. radiodurans.的代谢工程成为可能.

主要方法:

设计了一种单向量系统,用于表达I-E型级组件和crRNA.
利用CRISPR-Cascade系统击倒了phoN基因和基本的ssb基因.
针对ssb上游的监管元素 (RDRM) 来调节其表达.
通过同时使phoN和ssb沉默,证明了多个位置的准.

主要成果:

与II-A型dCas9系统相比,I-E型布系统对D.radiodurans表现出更好的耐受性.
实现了可测量phoN基因的显著淘汰 (10%的活性).
辐射抵抗基因ssb的失效导致辐射后恢复受损.
通过准RDRM.成功地防止了辐射时ssb表达的去压缩.
证明了 phoN 和 ssb 基因同时被淘汰.

结论:

开发的CRISPR-Cascade系统是D. radiodurans.中基因淘汰的有效工具.
这个系统有助于研究基本的基因,调节元件和辐射反应机制.
可编程的CRISPR干扰工具增强了D. radiodurans在工业生物技术和高通量研究中的潜力.

关键词:

克里斯普里是指克里斯普里.这是一个布式布式布式.迪诺科克斯 (Deinococcus radiodurans) 是一种可怕的植物.基因沉默是对基因进行沉默的方法.

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