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Controlling Gene Expression in Mammalian Cells Using Multiplexed Conditional Guide RNAs for Cas12a*
Lukas Oesinghaus1, Friedrich C Simmel1
1Physics Department, E14, TU Munich, Am Coulombwall 4a, 85748, Garching, Germany.
Angewandte Chemie (International Ed. in English)
|September 17, 2021
Summary
Researchers developed a new CRISPR-Cas12a system for precise gene editing. Conditional guide RNAs (gRNAs) are activated by cellular signals, enabling controlled gene regulation and potential therapeutic applications.
Area of Science:
- Molecular Biology
- Gene Editing Technologies
- Synthetic Biology
Background:
- CRISPR-associated (Cas) proteins offer powerful tools for gene manipulation.
- Precise spatiotemporal control over Cas protein activity is crucial for advanced research and therapies.
- Current methods for controlling Cas activity often lack modularity or require complex engineering.
Purpose of the Study:
- To develop a novel system for conditional activation of CRISPR-Cas12a activity in mammalian cells.
- To engineer guide RNAs (gRNAs) that can be processed and activated in response to specific cellular inputs.
- To create a compact, multi-input gene regulation system based on Cas12a and switchable gRNAs.
Main Methods:
- Transcribing conditional gRNAs for Cas12a using RNA polymerase II in mammalian cells.
- Implementing input-dependent processing of the gRNA 3' tail via RNA strand displacement, microRNA targeting, and ribozyme cleavage.
- Integrating Cas12a and multiple switchable gRNAs onto a single transcript using stabilizing RNA triplexes.
- Validating the system's functionality in HEK and mouse fibroblast cells using luminescence and fluorescence reporters, and endogenous gene upregulation.
Main Results:
- Demonstrated successful transcription and conditional activation of Cas12a by processed gRNAs in mammalian cells.
- Showcased diverse gRNA processing mechanisms, including strand displacement, microRNA-dependent, and ribozyme-mediated cleavage.
- Developed a single-transcript system integrating Cas12a with multiple independently switchable gRNAs using RNA triplexes.
- Confirmed the system's efficacy in controlling gene expression (luminescence, fluorescence) and upregulating an endogenous gene.
Conclusions:
- Conditional gRNAs for Cas12a can be engineered for input-dependent activation in mammalian cells.
- This platform enables the development of sophisticated, multi-input gene regulation systems.
- The developed technology holds promise for precise spatiotemporal control in basic research and therapeutic applications.
