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Controlling Gene Expression in Mammalian Cells Using Multiplexed Conditional Guide RNAs for Cas12a**
Angewandte Chemie International Edition ( IF 16.6 ) Pub Date : 2021-09-17 , DOI: 10.1002/anie.202107258
Lukas Oesinghaus 1 , Friedrich C Simmel 1
Affiliation  

Spatiotemporal control of the activity of CRISPR-associated (Cas) proteins is of considerable interest for basic research and therapeutics. Here, we show that conditional guide RNAs (gRNAs) for Cas12a can be transcribed in mammalian cells by RNA polymerase II, followed by activation via input-dependent processing of the 3′ tail of the gRNA transcript. We demonstrate processing using an RNA strand displacement mechanism, as well as microRNA-dependent processing, and cleavage by a guanine-responsive ribozyme. We further demonstrate that Cas12a along with several independently switchable gRNAs can be compactly integrated on a single transcript using stabilizing RNA triplexes, providing a route towards Cas12a-based gene regulation constructs with multi-input switching capabilities. The principle is shown to work in HEK and mouse fibroblast cells using luminescence, fluorescence, and is also demonstrated for the conditional upregulation of an endogenous gene.

中文翻译:

使用 Cas12a 的多重条件引导 RNA 控制哺乳动物细胞中的基因表达**

对 CRISPR 相关 (Cas) 蛋白活性的时空控制对基础研究和治疗具有重要意义。在这里,我们表明 Cas12a 的条件引导 RNA (gRNA) 可以通过 RNA 聚合酶 II 在哺乳动物细胞中转录,然后通过 gRNA 转录物的 3' 尾的输入依赖性处理激活。我们展示了使用 RNA 链置换机制的处理,以及依赖于 microRNA 的处理,以及鸟嘌呤响应核酶的切割。我们进一步证明 Cas12a 与几个独立的可切换 gRNA 可以使用稳定的 RNA 三链体紧密集成在单个转录本上,为具有多输入切换能力的基于 Cas12a 的基因调控构建体提供了途径。
更新日期:2021-10-19
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