Allosteric Conditional Guide RNAs for Cell-Selective CRISPR Control
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Solution Overview
Problem
Existing CRISPR/Cas systems face challenges in confining gRNA activity to a desired location and time within an organism, as gRNAs are constitutively active, necessitating additional measures for spatial and temporal control that can be complex and restrictive.
Innovation Solution
Development of allosteric conditional guide RNAs (cgRNAs) that change conformation in response to an RNA trigger, allowing for programmable and conditional control over Cas protein effector function, either activating or inactivating the target gene interaction based on the presence or absence of the trigger.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If gRNAs are made constitutively active to ensure continuous Cas protein effector function, then the CRISPR/Cas system maintains constant editing/silencing capability, but spatial and temporal control over gRNA activity is lost
Solution Approach 1:
The gRNA is designed with dynamic conformational changes that allow it to transition between active and inactive states based on cellular conditions. The gRNA structure includes a trigger-binding region that can bind to specific RNA triggers, causing conformational changes that activate or inhibit Cas protein effector function, thereby providing temporal control while maintaining reliability.
Solution Approach 2:
An RNA trigger acts as an intermediary molecule that mediates between the cell's regulatory signals and the gRNA's activity. The trigger binds to the gRNA's trigger-binding region, transmitting regulatory information that controls Cas protein effector function, enabling spatiotemporal control without direct protein intervention.
2Device complexity
If additional regulatory elements are added to control gRNA activity, then spatiotemporal control is improved, but the system complexity and potential off-target effects increase
Solution Approach 1:
The gRNA is segmented into distinct functional regions: a target-binding region, a trigger-binding region, and a Cas handle region. This segmentation allows independent optimization of each region's specificity and function, reducing off-target effects while maintaining control capability. The trigger-binding region can be designed to be highly specific to avoid unintended activation.
Solution Approach 2:
The system utilizes changes in molecular parameters (conformational state, binding affinity) to control gRNA activity. By adjusting the thermodynamic parameters of the trigger-binding interaction, the system achieves precise control over Cas protein effector function activation, minimizing off-target effects through parameter optimization rather than adding complex regulatory circuits.
3Adaptability or versatility
If the gRNA structure is modified to include trigger-binding regions, then conditional control is enabled, but the gRNA's ability to bind target genes may be affected
Solution Approach 1:
The gRNA is designed with local quality differentiation: the target-binding region maintains high complementarity to the target gene for reliable binding, while the trigger-binding region has specialized sequences for conditional control. The Cas handle region is optimized for Cas protein interaction. This local optimization ensures that adding trigger-binding capability does not compromise target binding efficiency.
Solution Approach 2:
The gRNA regulation is moved to another dimension by adding a trigger-binding region that responds to RNA triggers. This creates a new layer of control where the gRNA's activity is dimensionally expanded from simple target binding to conditional activation based on trigger presence, maintaining target binding reliability through spatial separation of functions.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables precise spatiotemporal regulation of Cas protein effector functions, such as editing, silencing, or binding, by independently controlling the regulatory scope and target through cgRNA mechanisms, enabling precise control over the application and application of the CRISPR/Cas system.
Implementation Method 1
upon hybridization to the cognate RNA trigger, the cgRNA is inactivated
Implementation Method 2
allosteric conditional guide RNAs (cgRNAs) that change conformation in response to an RNA trigger
Data Source
AI summary
Programmable guide RNAs (gRNAs) play a central role in the CRISPR revolution sweeping biology and medicine by directing the function of a Cas protein effector to a target gene of choice. To achieve programmable control over regulatory scope, the activity of a conditional guide RNA (cgRNA) depends on the presence or absence of an RNA trigger, allowing for cell-selective regulation of CRISPR/Cas function. Unlike a standard gRNA, a cgRNA is programmable at multiple levels, with the target-binding sequence controlling the target of Cas activity (edit, silence, induce, or bind a gene of choice) and the triggerbinding sequence controlling the scope of Cas activity. cgRNA mechanisms that are allosteric allow for independent design of the target and trigger sequences, providing the flexibility to select the regulatory target and scope independently. Disclosed herein are allosteric cgRNA mechanisms for both ON→OFF logic (conditional inactivation by an RNA trigger) and OFF→ON logic (conditional activation by an RNA trigger). Allosteric cgRNAs enable restriction of CRISPR/Cas function to a desired cell type, tissue, organ, or disease state. Allosteric cgRNAs provide a versatile platform for cell-selective and tissue-selective research tools, biotechnologies, diagnostics, and therapeutics.