Zinc Finger Degradation Domains for CRISPR Temporal Control
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Solution Overview
Problem
Current genome editing technologies, such as CRISPR-Cas systems, lack precise and switchable control over effector activities, leading to issues like elevated off-target DNA cleavage and the need for methods to regulate CRISPR effector protein levels and activity duration.
Innovation Solution
Development of hybrid zinc finger polypeptides optimized for degradation by immunomodulatory drugs like pomalidomide, avadomide, and lenalidomide, which can be integrated into CRISPR-Cas proteins to control their activity and induce proteasomal degradation, allowing for precise regulation of genome editing outcomes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If CRISPR effector proteins are used at high concentrations to enhance genome editing efficiency, then productivity is improved, but off-target DNA cleavage increases reducing reliability
Solution Approach 1:
The patent applies dynamics by making the CRISPR effector protein concentration time-dependent through controlled degradation. The system transitions from high concentration (for efficient on-target editing) to low concentration (to minimize off-target effects) automatically over time, resolving the contradiction between productivity and reliability
Solution Approach 2:
The patent changes the concentration parameter of the CRISPR effector protein over time through degradation control. By modifying the protein half-life parameter, the system achieves high initial concentration for efficient editing followed by natural decline to reduce off-target effects, simultaneously improving both productivity and reliability
2Duration of action of moving object
If CRISPR effector proteins are maintained for extended periods to ensure complete genome modification, then duration of action is improved, but off-target effects increase reducing safety
Solution Approach 1:
The patent implements periodic action through controlled degradation and potential re-synthesis cycles of the CRISPR effector protein. The protein is maintained at active levels only for the necessary duration to achieve editing, then degraded to eliminate prolonged off-target effects, balancing duration of action with safety
Solution Approach 2:
The system dynamically adjusts the duration of CRISPR effector protein activity through degradation control mechanisms. The protein persists long enough to complete necessary edits but is subsequently removed to prevent extended off-target effects, resolving the contradiction between adequate duration and safety
3Reliability
If degradation control mechanisms are added to CRISPR systems to improve precision, then reliability is improved, but device complexity increases
Solution Approach 1:
The patent applies self-service by utilizing the cell's endogenous proteasomal degradation system to control CRISPR effector protein levels. The degradation tags recruit existing cellular machinery (E3 ligases, proteasome) to degrade the effector protein, achieving precision control without adding complex external degradation systems, thus improving reliability while minimizing increased 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 finely tuned control over CRISPR effector activities, reducing off-target effects and allowing for temporal regulation of genome editing by degrading the Cas proteins after desired modifications, thereby enhancing the precision and safety of genome editing processes.
Implementation Method 1
Exploitation of a mechanism to target proteins for degradation in the proteasome would be one approach to degrade Cas effector protein after its use
Implementation Method 2
Typically, once proteins are no longer needed in a cell, they are tagged in the cell with ubiquitin utilizing an E3 ligase to designate the protein for degradation in the proteasome
Data Source
AI summary
The disclosure includes compositions comprising synthetic zinc finger degrons, and their use with non-naturally occurring or engineered programmable nucleases. Compositions specifically targeting the engineered programmable nucleases for control of gene editing outcomes, and compositions, systems and method of use are further detailed.