Optochemical CRISPR-Cas9 Control for Off-Target Cleavage
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Solution Overview
Problem
Current genome editing technologies lack precise and switchable methods to regulate CRISPR effector activities across dimensions of dose, target, and time, leading to elevated off-target DNA cleavage and inefficient control of gene expression.
Innovation Solution
Development of optochemical CRISPR systems with destabilizing domains that bind to photocaged ligands upon light exposure, allowing rapid and dosable activation of CRISPR activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If CRISPR effector proteins are expressed at high concentrations to enhance genome editing efficiency, then productivity is improved, but off-target DNA cleavage increases reducing reliability
Solution Approach 1:
The system performs preliminary action by pre-expressing the CRISPR effector protein in an inactive, degraded state before light exposure. The destabilization domain ensures the protein is prepared and ready but kept inactive until the precise moment of light activation, preventing premature off-target effects while ensuring immediate availability for on-target editing when needed.
Solution Approach 2:
The system implements dynamics by making the CRISPR effector protein's stability and activity dynamically controllable through light exposure. The protein transitions from an unstable, degraded state to a stable, active state in response to light, allowing real-time adjustment of protein levels and activity to optimize both editing efficiency and specificity without permanent high concentration expression.
2Duration of action of stationary object
If CRISPR effector activity is continuously active to maintain gene expression control, then duration of action is improved, but ability to rapidly reverse activity is lost worsening adaptability
Solution Approach 1:
The system implements periodic action through light-exposure cycles. The CRISPR effector protein can be activated by light exposure to induce gene expression changes, then deactivated by removing light exposure, allowing the protein to be degraded. This enables repeated cycles of activation and deactivation, providing both sustained control during light exposure and rapid reversibility when light is removed, enhancing both duration of action and adaptability.
3Device complexity
If conventional CRISPR systems are used without optochemical control to simplify the system, then device complexity is reduced, but precision and switchability of CRISPR activity control deteriorates
Solution Approach 1:
The system introduces an intermediary element - the light-responsive destabilization domain - that mediates between the simple light stimulus and the complex CRISPR effector activity. This intermediary allows precise temporal and spatial control of protein stability through light exposure, enabling high-precision switchable control without requiring complex regulatory circuits or multiple protein components, thus achieving precision enhancement with minimal added complexity.
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 and rapid control of CRISPR activity, reducing off-target effects and enabling fine-tuned gene expression through light-activated ligand binding.
Implementation Method 1
one or more photocaged ligands specific for the one or more destabilizing domains, wherein the ligand can bind to the one or more destabilizing domains upon exposure to light
Data Source
AI summary
The disclosure includes non-naturally occurring or engineered DNA- or RNA-guided nuclease systems, comprising CRISPR enzymes associated with at least one destabilization domain (DD) and photocaged stabilization ligands with at least one photocage molecule, along with compositions, systems and complexes involving such systems, nucleic acid molecules and vectors encoding the same, delivery systems involving the same, uses thereof.