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Spatial and temporal control of gene manipulation in Drosophila via drug-activated Cas9 nucleases.
Insect Biochemistry and Molecular Biology ( IF 3.8 ) Pub Date : 2020-02-24 , DOI: 10.1016/j.ibmb.2020.103336
Nhan Huynh 1 , Song Wang 1 , Kirst King-Jones 1
Affiliation  

Advances in CRISPR/Cas9 have revolutionized molecular biology and greatly facilitated the ability to manipulate gene function through the creation of precisely engineered mutants. We recently reported a collection of modular gateway-compatible Cas9/gRNA Drosophila lines to interfere with gene expression in a tissue-specific manner, including polytene tissues. However, most current in vivo CRISPR/Cas9 tools cannot temporally control the induction of Cas9 or gRNAs via external stimuli such as RU486. A drug-inducible CRISPR/Cas9 system would allow studying genes at later stages where early lethality is an issue. This would be especially useful when combined with tissue-specific expression of Cas9 or gRNAs, allowing for full spatiotemporal control. Here, we present a RU486-inducible version of Cas9 and also show that a Rapamycin-inducible Cas9, previously used in mammalian cell culture, works in Drosophila as well. Both RU486 and rapamycin-inducible Cas9 work in vivo and in Drosophila cell culture. We also present split Cas9 constructs for rapamycin-dependent gene disruption and activation. These approaches establish drug-inducible and thus temporally controlled CRISPR/Cas9 tools for gene disruption and expression in a living model organism. Our CRISPR/Cas9 vector collection can be easily adapted for any tissue and provides higher fidelity compared to RNAi approaches.

中文翻译:

通过药物激活的Cas9核酸酶在果蝇中进行基因操作的时空控制。

CRISPR / Cas9的进步彻底改变了分子生物学,并通过创建精确设计的突变体大大促进了操纵基因功能的能力。我们最近报道了模块化网关兼容Cas9 / gRNA果蝇系的集合,以组织特异性方式(包括多烯组织)干扰基因表达。然而,目前大多数的体内CRISPR / Cas9工具无法通过外部刺激(例如RU486)暂时控制Cas9或gRNA的诱导。药物诱导的CRISPR / Cas9系统将允许在较早致死性的后期研究基因。当与Cas9或gRNA的组织特异性表达结合使用时,这将特别有用,从而可以进行完全的时空控制。在这里,我们介绍了RU486诱导型的Cas9,并显示了雷帕霉素诱导的Cas9,以前用于哺乳动物细胞培养,也可用于果蝇。RU486和雷帕霉素诱导的Cas9均可在体内和果蝇细胞培养物中发挥作用。我们还介绍了雷帕霉素依赖性基因破坏和激活的拆分Cas9构建体。这些方法建立了可药物诱导的,因此受到时间控制的CRISPR / Cas9工具,用于在生物模型生物中进行基因破坏和表达。与RNAi方法相比,我们的CRISPR / Cas9载体集合可轻松适应任何组织,并提供更高的保真度。这些方法建立了可药物诱导的,因此受到时间控制的CRISPR / Cas9工具,用于在生物模型生物中进行基因破坏和表达。与RNAi方法相比,我们的CRISPR / Cas9载体集合可轻松适应任何组织,并提供更高的保真度。这些方法建立了可药物诱导的,因此受到时间控制的CRISPR / Cas9工具,用于在生物模型生物中进行基因破坏和表达。与RNAi方法相比,我们的CRISPR / Cas9载体集合可轻松适应任何组织,并提供更高的保真度。
更新日期:2020-02-25
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