Activatable Cas9 Proteins for Spatial and Temporal Control
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Solution Overview
Problem
Current CRISPR/Cas9 systems lack precise spatial and temporal control, leading to off-target effects and inefficient gene editing in specific tissues and time points.
Innovation Solution
Engineering light-activated and chemical-activated Cas9 proteins with site-specifically installed caged lysine or chemically activatable lysine residues, allowing for optical or chemical control of Cas9 function, enabling precise spatial and temporal control of gene editing and expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If CRISPR/Cas9 system is used for gene editing, then gene editing efficiency is improved, but off-target effects increase due to lack of spatial and temporal control
Solution Approach 1:
The patent applies preliminary action by expressing the Cas9 protein and guide RNA in the cell beforehand, but keeping the Cas9 protein in an inactive state through chemical caging. The system is prepared and positioned at the target location, but the actual cutting action is delayed until the activator is administered. This allows the editing machinery to be in place before activation, improving efficiency while preventing premature off-target effects.
Solution Approach 2:
The patent uses a chemical activator as an intermediary substance that triggers the conversion of inactive caged Cas9 to active Cas9. This intermediary mediates between the prepared editing system and the actual gene cutting action, providing precise temporal control. The activator enables the system to transition from a harmless prepared state to an active editing state only when needed.
2Adaptability or versatility
If CRISPR/Cas9 system is activated in all tissues, then gene editing can be performed throughout the organism, but precision of targeting specific tissues is lost
Solution Approach 1:
The patent applies local quality by making the Cas9 protein activity dependent on local administration of the chemical activator. Different tissues can be targeted by administering the activator to specific locations, creating spatially differentiated editing activity. The same Cas9 protein can remain inactive in most tissues while being activated locally where the activator is present, achieving precise tissue-specific targeting.
Solution Approach 2:
The system allows preliminary expression and distribution of inactive Cas9 protein throughout the organism without causing harm. The protein is prepared in advance and positioned in all tissues, but remains inert until local activation. This preliminary action enables rapid response when activator is administered to specific tissues, achieving both versatility and precision.
3Duration of action of stationary object
If CRISPR/Cas9 is administered continuously, then sustained gene editing activity is maintained, but temporal control and reduction of prolonged off-target effects are compromised
Solution Approach 1:
The patent enables periodic or pulsed activation of Cas9 activity through repeated administration of the chemical activator. Instead of continuous activity, the system can be activated in controlled pulses at specific time points. This provides precise temporal control while maintaining sustained editing capability through multiple activation cycles, reducing prolonged off-target effects between pulses.
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
This approach allows for conditional control of gene editing, reducing off-target effects and enabling targeted gene activation or repression in specific tissues and time points, enhancing the precision of CRISPR/Cas9 applications.
Implementation Method 1
light-activated and chemical-activated Cas9 proteins were engineered through the site-specific installation of a caged lysine
Implementation Method 2
chemically-activatable lysine, respectively. Specific lysine residues were identified as activatable sites that can be modified to optically or chemically control Cas9 function
Data Source
AI summary
Disclosed herein is a genetically encoded light- or chemically-activated Cas9 engineered through the site-specific installation of an activatable lysine amino acid. Such activatable Cas9 proteins can be used in CRISPR/Cas9 systems to control gene expression temporally, spatially, or both. Systems, methods, kits, and compositions for manipulation of sequences and/or activities of target sequences are provided. Also provided are methods of directing CRISPR complex formation in cells and methods for selecting specific cells by introducing precise mutations utilizing the CRISPR/Cas9 system.