Modular CRISPR Transcriptional Activators Enabled by RNA-Sensing Guide RNAs in Mammalian Cells and Zebrafish Embryos

Core Concepts

Engineered RNA-sensing CRISPR guide RNAs (iSBH-sgRNAs) enable conditional activation of CRISPR transcriptional regulators in response to specific RNA sequences in mammalian cells and zebrafish embryos.

Abstract

The authors present a system for modulating CRISPR activity in response to RNA detection using engineered single-guide RNAs (iSBH-sgRNAs). The iSBH-sgRNAs are designed to fold into complex secondary structures that inhibit their activity in the absence of complementary RNA triggers. When the iSBH-sgRNAs detect their target RNA sequences, the structures unfold, activating the CRISPR system and enabling transcriptional regulation.
The authors first demonstrate that first-generation iSBH-sgRNA designs can detect short RNA triggers and activate CRISPR transcriptional activators (dCas9-Vp64) in HEK293T cells. They then optimize the designs to enable detection of longer RNA triggers up to 300 nucleotides.
To further improve the system, the authors develop the MODesign computational pipeline, which allows for the independent design of the CRISPR target sequence and the RNA-sensing component. This modular approach enables detection of a wider range of RNA sequences.
Mechanistic investigations reveal that the iSBH-sgRNA activation involves cleavage of the engineered sgRNA components, likely by cellular double-stranded RNA processing pathways. The authors identify key positions for chemical modifications to stabilize the iSBH-sgRNAs and demonstrate their functionality in zebrafish embryos.
The authors discuss the potential of this RNA-sensing CRISPR technology for developing more specific and effective gene editing approaches, as well as therapeutic applications that leverage endogenous RNA biomarkers to control CRISPR activity.

Stats

The authors report that the presence of complementary RNA triggers leads to a 3.2-fold increase in the number of zebrafish embryos displaying high ECFP expression compared to the absence of triggers.

Quotes

"Our results show that iSBH-sgRNAs are functional in developing zebrafish embryos. Furthermore, gaining insight into the mechanism of the iSBH-sgRNA activation has allowed us to identify critical positions for chemical modifications that stabilised our design."
"In the pursuit of targeted gene editing, identifying cell surface biomarkers can be a daunting task for certain cell types or diseases. However, a promising solution may lie in utilizing endogenous RNA biomarkers to activate CRISPR activity."

Key Insights Distilled From

Specific Modulation of CRISPR Transcriptional Activators through RNA-Sensing Guide RNAs in Mammalian Cells and Zebrafish Embryos

by Pelea,O., Ma... at www.biorxiv.org 05-08-2023

https://www.biorxiv.org/content/10.1101/2023.05.08.539738v2

Deeper Inquiries

What other types of endogenous RNA biomarkers could be leveraged as triggers for the iSBH-sgRNA system, and how could this expand its therapeutic applications?

The iSBH-sgRNA system could potentially leverage various endogenous RNA biomarkers as triggers, expanding its therapeutic applications significantly. One type of RNA biomarker that could be utilized is long non-coding RNAs (lncRNAs). These lncRNAs play crucial roles in gene regulation and have been implicated in various diseases. By designing iSBH-sgRNAs to detect specific lncRNAs associated with certain diseases, such as cancer or neurodegenerative disorders, the system could enable precise targeting of affected cells for therapeutic interventions.
Moreover, microRNAs (miRNAs) could also serve as valuable triggers for the iSBH-sgRNA system. MiRNAs are small non-coding RNAs that regulate gene expression post-transcriptionally and are dysregulated in many diseases. By designing iSBH-sgRNAs to detect disease-specific miRNAs, the system could be used to modulate gene expression in a highly targeted manner, offering potential therapeutic benefits.
Furthermore, viral RNAs could be targeted by the iSBH-sgRNA system for antiviral applications. By designing iSBH-sgRNAs to detect viral RNA sequences, the system could be used to activate CRISPR-mediated antiviral responses in infected cells, providing a novel approach to combat viral infections.

How could the iSBH-sgRNA system be further optimized to achieve even tighter control over CRISPR activity and reduce off-target effects in vivo?

To achieve tighter control over CRISPR activity and reduce off-target effects in vivo, the iSBH-sgRNA system could be further optimized in several ways:

Enhanced Trigger Specificity: By refining the design of iSBH-sgRNAs to have higher specificity for RNA triggers, the system can minimize off-target effects and ensure precise activation only in the presence of the intended RNA biomarkers.

Improved Chemical Modifications: Implementing advanced chemical modifications in iSBH-sgRNAs to enhance stability and prevent degradation by cellular nucleases can improve the overall efficiency and specificity of the system.

Optimized Delivery Methods: Developing more efficient delivery methods for iSBH-sgRNAs to ensure uniform distribution and uptake in target tissues can enhance the system's effectiveness and reduce variability in activation levels.

Fine-Tuning CRISPR Components: Adjusting the expression levels of CRISPR effectors and reporters, as well as optimizing the choice of CRISPR activators, can help fine-tune the system for optimal performance and minimize off-target effects.

Incorporating Feedback Mechanisms: Implementing feedback mechanisms that monitor and regulate CRISPR activity based on the level of RNA triggers detected can provide dynamic control over gene editing processes, further reducing off-target effects.

What potential applications beyond gene editing and transcriptional regulation could this RNA-sensing CRISPR technology enable, such as in the fields of synthetic biology or cellular reprogramming?

The RNA-sensing CRISPR technology could open up a wide range of applications beyond gene editing and transcriptional regulation, particularly in the fields of synthetic biology and cellular reprogramming:

RNA-Based Logic Circuits: The ability of iSBH-sgRNAs to detect specific RNA triggers could be harnessed to design intricate RNA-based logic circuits for synthetic biology applications, enabling the development of complex cellular computing systems.

RNA-Triggered Drug Delivery: By coupling the iSBH-sgRNA system with drug delivery mechanisms, researchers could create RNA-triggered drug release systems that activate therapeutic compounds only in the presence of specific RNA biomarkers, offering targeted and controlled drug delivery.

Cell Fate Reprogramming: Leveraging the RNA-sensing capabilities of iSBH-sgRNAs, researchers could explore novel approaches to cellular reprogramming by activating specific gene expression patterns in response to endogenous RNA signals, facilitating the conversion of cell types for regenerative medicine applications.

RNA-Based Biosensors: The iSBH-sgRNA system could be adapted to serve as a platform for developing RNA-based biosensors that detect and respond to specific RNA signatures, enabling real-time monitoring of cellular processes and disease biomarkers.

Environmental Sensing: By engineering iSBH-sgRNAs to detect environmental RNA triggers, such as pathogen-specific RNA sequences or pollutant-induced RNA changes, the technology could be applied in environmental monitoring and biosafety applications.