Real-time sensing and control of cellular responses by an RNA-sensing CRISPR/dCas9 engineered system
Date
2021
Authors
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Publisher
University of Delaware
Abstract
To effectively reprogram cell regulatory networks and manipulate mammalian host cells towards desired phenotypes, it is critical to have technology platforms that provide precise and accurate targeting of genes. The use of CRISPR interference (CRISPRi) or CRISPR activation (CRISPRa) for targeted silencing or upregulation of transcription, respectively, is currently one of the most utilized technologies for predictable control over gene expression. The CRISPR system allows for sequence-specific targeting of genes but inherently lacks the ability to incorporate useful endogenous signals for spatiotemporal control of gene expression. Here we present the design and further characterization of a class of riboregulators through the incorporation of toehold riboswitches into sgRNA scaffolds, which would provide an additional level of inducible cellular control. ☐ This artificial circuit is able to detect the presence of specific RNA and switch on transcriptional level gene regulation through RNA-RNA strand displacement reactions, which are governed by predictable Watson-Crick base pairing. These synthetic constructs can be programmed to process specific information within the cell including changes in native metabolism and stress responses. We demonstrate the programmability and adaptability of these engineered systems to control gene expression in mammalian systems with minimal infidelity. ☐ This system can be engineered to regulate a reporter protein, with a conditional response greater than 30-fold as compared to the control. We show the robustness of our system can be adapted to other sequences with high levels of success. We show the ability to create AND gate architecture when we combine two different RNA-sensing CRISPR/Cas9 systems to show the combined response for both microRNAs (miRs) and full-length mRNAs. We also demonstrate the ability to create a cascade-type system to control protein degradation. We apply multiple RNA-sensing approaches to show up to 8-fold reporter knockdown for protein degradation applications. ☐ We envision these synthetic riboregulators can be applied to host cells for improved cellular control towards both growth or product quality attributes. We also envision this technology can be used to probe disease-states and developmental stages that demonstrate dynamic expression of RNAs to be used as inputs. This technology has the potential to function as an RNA-based master regulator for autonomous cellular control to direct specific phenotype in mammalian cells.
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Keywords
CRISPR/dCas9, Gene regulation, Protein degradation, RNA, thgRNA