Allosteric Conditional Guide RNAs for Cell-Selective CRISPR/Cas 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, necessitating additional regulatory pathways that may limit spatial and temporal control.
Innovation Solution
Development of allosteric conditional guide RNAs (cgRNAs) that incorporate a target-binding region and a trigger-binding region, allowing for conditional activation or inactivation in response to a cognate RNA trigger, enabling independent control over regulatory scope and target selection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If gRNA activity is made constitutively active to enable versatile programmable regulation, then the functional versatility of CRISPR/Cas system is improved, but the ability to confine gRNA activity to desired location and time deteriorates
Solution Approach 1:
The gRNA is segmented into distinct functional regions: a target-binding region for gene-specific activity and a trigger-binding region for conditional activation. This segmentation allows independent optimization of target specificity and temporal/spatial control through the trigger mechanism.
Solution Approach 2:
The gRNA transitions from a static constitutively active state to a dynamic conditionally active state. The trigger-binding region enables the gRNA to switch between active and inactive states based on trigger presence, providing temporal and spatial control over CRISPR/Cas function.
2Ease of operation
If additional regulatory pathways are introduced to achieve spatiotemporal control, then the ability to confine gRNA activity is improved, but the device complexity increases
Solution Approach 1:
The trigger-binding region is merged directly into the gRNA structure, combining the regulatory function with the guide RNA itself. This integration eliminates the need for separate regulatory pathways and proteins, reducing system complexity while maintaining spatiotemporal control.
Solution Approach 2:
The trigger-binding region serves multiple functions: it provides conditional activation, enables spatiotemporal control, and maintains compatibility with existing CRISPR/Cas effectors. This multi-functionality reduces the need for additional specialized components.
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
cgRNAs provide programmable and spatiotemporal control over CRISPR/Cas function, allowing for cell-selective and tissue-selective regulation without relying on additional regulatory pathways, facilitating applications in research, diagnostics, and therapeutics.
Implementation Method 1
upon hybridization to the cognate RNA trigger, the cgRNA is inactivated
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 trigger-binding 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.