Native CRISPR-Cas-based programmable multiplex gene repression in Klebsiella variicola

IF 4.6 Q2 MATERIALS SCIENCE, BIOMATERIALS
Zhifeng Mo, Siying Lin, Ting Li, Guohui Yu, Yunhao Sun, Jianuan Zhou, Zeling Xu
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Abstract

Klebsiella variicola is a Gram-negative bacterium that is frequently isolated from a wide variety of natural niches. It is a ubiquitous opportunistic pathogen that can cause diverse infections in plants, animals, and humans. It also has significant biotechnological potential. However, due to the lack of efficient genetic tools, the molecular basis contributing to the pathogenesis and beneficial activities of K. variicola remains poorly understood. In this study, we found and characterized a native type I-E CRISPR-Cas system in a recently isolated K. variicola strain KV-1. The system cannot cleave target DNA sequences due to the inactivation of the Cas3 nuclease by a transposable element but retains the activity of the crRNA-guided Cascade binding to the target DNA sequence. A targeting plasmid carrying a mini-CRISPR to encode a crRNA was designed and introduced into the KV-1 strain, which successfully repurposed the native type I-E CRISPR-Cas system to inhibit the expression of the target gene efficiently and specifically. Moreover, by creating a mini-CRISPR to encode multiple crRNAs, multiplex gene repression was achieved by providing a single targeting plasmid. This work provides the first native CRISPR-Cas-based tool for programmable multiplex gene repression in K. variicola, which will facilitate studying the pathogenic mechanism of K. variicola and enable metabolic engineering to produce valuable bioproducts.

Abstract Image

变异克雷伯氏菌中基于 CRISPR-Cas 的原生可编程多重基因抑制
变异克雷伯氏菌是一种革兰氏阴性细菌,经常从各种自然环境中分离出来。它是一种无处不在的机会性病原体,可引起植物、动物和人类的各种感染。它还具有巨大的生物技术潜力。然而,由于缺乏有效的遗传工具,人们对 K. variicola 的致病机理和有益活动的分子基础仍然知之甚少。在本研究中,我们发现并鉴定了最近分离出的 K. variicola 菌株 KV-1 中的原生 I-E 型 CRISPR-Cas 系统。由于转座元件使Cas3核酸酶失活,该系统不能切割目标DNA序列,但保留了crRNA引导的Cascade与目标DNA序列结合的活性。我们设计了一种携带微型CRISPR以编码crRNA的靶向质粒,并将其导入到KV-1菌株中,成功地将原生的I-E型CRISPR-Cas系统重新利用,从而高效、特异地抑制了靶基因的表达。此外,通过创建一个迷你 CRISPR 来编码多个 crRNA,只需提供一个靶向质粒就能实现多重基因抑制。这项工作提供了第一个基于CRISPR-Cas的可编程多重基因抑制K. variicola的原生工具,这将有助于研究K. variicola的致病机制,并通过代谢工程生产有价值的生物产品。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
ACS Applied Bio Materials
ACS Applied Bio Materials Chemistry-Chemistry (all)
CiteScore
9.40
自引率
2.10%
发文量
464
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