CRISPR Guide Adaptor Destabilization Domains for Off-Target Control
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Solution Overview
Problem
Current genome editing technologies lack methods for precise, switchable regulation of CRISPR effector activities across multiple dimensions, including dose, target, and time, with high concentrations leading to elevated off-target DNA cleavage and a need for improved control of gene expression.
Innovation Solution
A fusion protein comprising destabilization domains (DD) and adaptor proteins that bind to guide RNA or DNA, allowing for better control of CRISPR/Cas systems, reducing off-target events and enabling conditional or inducible activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If high concentrations of CRISPR effector proteins are used to enhance genome editing efficiency, then productivity is improved, but off-target DNA cleavage increases causing harmful effects
Solution Approach 1:
The patent applies dynamics by making CRISPR effector protein expression conditional and temporally controllable through destabilization domains. The system transitions from static high-expression to dynamic controlled-expression, allowing effector proteins to be present only when and where needed, thus maintaining high editing efficiency while minimizing off-target effects through reduced background expression.
Solution Approach 2:
The patent changes the stability parameter of CRISPR effector proteins by fusing them to destabilization domains (DD). This parameter change allows precise control over protein half-life and concentration levels, enabling the system to achieve high productivity when needed while rapidly reducing harmful off-target activity when the destabilizing ligand is removed or degraded.
2Duration of action of stationary object
If constitutive expression of CRISPR effector proteins is used to maintain continuous activity, then duration of action is improved, but precise temporal control is lost
Solution Approach 1:
The patent implements feedback control through destabilization domains that respond to the presence or absence of stabilizing ligands. The system continuously monitors and adjusts effector protein levels based on ligand availability, providing precise temporal control while maintaining the ability for prolonged activity when ligand is present. This feedback mechanism replaces constitutive expression with regulated expression.
Solution Approach 2:
The system transforms static constitutive expression into dynamic inducible expression. The CRISPR effector proteins are now present only during specific time windows when stabilizing ligand is provided, enabling precise temporal control for applications such as sequential editing or synchronized cellular states while maintaining continuous activity during the desired window.
3Reliability
If high levels of CRISPR effector proteins are expressed to ensure sufficient target coverage, then reliability is improved, but off-target effects increase causing harmful factors
Solution Approach 1:
The patent changes the concentration parameter of CRISPR effector proteins from high constitutive levels to controlled transient levels. By using destabilization domains, the system achieves sufficient target coverage during the active window when ligand is present, while rapidly clearing proteins to eliminate off-target effects. This parameter control maintains reliability without the harmful consequences of sustained high expression.
4Ease of manufacture
If simple CRISPR effector expression is used to maintain device simplicity, then ease of manufacture is improved, but regulatory control capability is reduced
Solution Approach 1:
The patent merges the CRISPR effector protein with a destabilization domain to create a single fusion protein construct. This combination integrates both the genome editing function and the regulatory control mechanism into one element, maintaining ease of manufacture through single-vector delivery while gaining sophisticated temporal and dosage control capabilities. The fusion protein approach simplifies the system compared to separate expression components.
Data Source
AI summary
The disclosure includes non-naturally occurring or engineered DNA- or RNA-guided nuclease systems, comprising guide-binding adaptors each associated with at least one destabilization domain (DD), along with compositions, systems and complexes involving such systems, nucleic acid molecules and vectors encoding the same, delivery systems involving the same, uses therefor.