在Fusobacterium nucleatum中使用CRISPR干扰进行高效和快速的基因失活
Peng Zhou1, Bibek G C1, Flynn Stolte1
1Department of Microbiology & Molecular Genetics, The University of Texas Health Science Center, Houston, Texas, USA.
Applied and environmental microbiology
|January 8, 2024
概括
我们开发了一种CRISPR干扰 (CRISPRi) 系统,可以轻松地使Fusobacterium物种中的基因失活. 这种工具简化了遗传研究,并使以前难以处理的菌株的功能分析成为可能.
科学领域:
- 微生物学 微生物学
- 分子生物学分子生物学
- 遗传学 是一个遗传学.
背景情况:
- 在 * Fusobacterium nucleatum * 中的基因功能研究是具有挑战性的,因为难以激活基因和菌株的遗传固性.
- 对于许多fusobacterial菌株来说,传统的基因删除方法耗时且往往效率低下.
研究的目的:
- 开发和验证一种基于 рибо开关的可诱导CRISPR干扰 (CRISPRi) 系统,用于有效地对*Fusobacterium*物种进行基因沉默.
- 为了证明该系统在不同的 * Fusobacterium * 菌株中适用,包括临床上相关的和以前难以治疗的菌株.
主要方法:
- 利用由单导 RNA (sgRNA) 引导的核酶非活性 Cas9 (dCas9) 来抑制目标基因转录.
- 采用一种可诱导神素的核糖开关来控制dCas9的表达.
- 应用CRISPRi系统研究非必需基因 (*ftsX*, *radD*),必需基因 (*bamA*, *ftsZ*),以及参与各种 *Fusobacterium* 菌株代谢途径 (*tnaA*, *clpB*) 的基因.
主要成果:
- 成功抑制了目标基因,导致可观察到的表型,如丝状细胞形成 (*ftsX*),废除凝聚 (*radD*),破坏了膜完整性 (*bamA*) 和延长细胞 (*ftsZ*).
- 在 *F. nucleatum* 临床菌株 CTI-2 和 *Fusobacterium periodonticum* 中证明了基因沉默,降低了醇合成 (*tnaA*) 和增加了热敏度 (*clpB*).
- 克里斯皮尔系统在多种菌菌株中被证明是有效的,简化了基因功能调查.
结论:
- 开发的可诱导 рибо开关的CRISPRi系统为*Fusobacterium*物种的基因失活提供了一个强大而多功能工具.
- 这个系统克服了以前的局限性,使得在广泛的菌菌株中实现了高效的基因操纵和功能基因组学.
- 该技术有助于深入研究fusobacterial病原体,并可能有助于制定有针对性的治疗策略.
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