期刊
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
卷 60, 期 44, 页码 23894-23902出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/anie.202107258
关键词
molecular computing; mRNA; RNA structures; synthetic biology; toehold-mediated strand displacement
资金
- Deutsche Forschungsgemeinschaft (DFG) [SI761/5-1]
- European Research Council (ERC) [694410]
- Max Planck School Matter to Life
- Projekt DEAL
The study demonstrates that conditional guide RNAs (gRNAs) for Cas12a can be transcribed in mammalian cells by RNA polymerase II and activated via input-dependent processing of the 3' tail of the gRNA transcript, providing a new route towards Cas12a-based gene regulation constructs. The principle has been shown to work in HEK and mouse fibroblast cells using luminescence, fluorescence, and for the conditional upregulation of an endogenous gene.
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.
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