Controlling Gene Expression in Mammalian Cells Using Multiplexed Conditional Guide RNAs for Cas12a. Issue 44 (29th September 2021)

• Record Type: Journal Article
• Authors: Oesinghaus, Lukas; Simmel, Friedrich C.
• Is Part Of: Angewandte Chemie international edition. Volume 60:Issue 44(2021)
• Page Start: 23894
• Page End: 23902
• Publication Date: 2021-09-29
• Subjects: molecular computing -- mRNA -- RNA structures -- synthetic biology -- toehold-mediated strand displacement; Chemistry -- Periodicals; 540
• DOI: 10.1002/anie.202107258
• Languages: English
• ISSNs: 1433-7851

Abstract

Abstract: 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. Abstract : Conditional processing of Pol II‐transcribed Cas12a gRNAs for gene activation using three different activation modalities was achieved: RISC‐based cleavage, an aptazyme, and an RNA strand displacement mechanism. Several switchable guide RNAs along with Cas12a itself can be integrated on a single transcript using a stabilizing triplex motif.