Ligand-Dependent Intein Cas9 Control for Off-Target Activity
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Solution Overview
Problem
Existing RNA-guided nucleases, such as Cas9, exhibit significant off-target activity, leading to cellular toxicity and undesired genomic alterations, limiting their suitability for clinical and research applications.
Innovation Solution
Development of Cas9 proteins fused with ligand-dependent inteins that inhibit enzymatic activities in the absence of a ligand, allowing controlled activation upon ligand binding to minimize off-target effects.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If RNA-guided nucleases (Cas9) are used for genome engineering, then targeted DNA cleavage capability is achieved, but off-target activity increases leading to cellular toxicity
Solution Approach 1:
The intein is inserted into the Cas9 protein structure in advance, creating a self-excising domain that will be activated under specific conditions. This preliminary structural modification allows the Cas9 protein to remain inactive during delivery and expression, then become activated only when the intein excises itself in response to specific cellular conditions, thereby preventing off-target activity before it can occur
Solution Approach 2:
The intein acts as an intermediary element between the Cas9 nuclease and its target DNA. This self-excising domain serves as a conditional mediator that controls the activation state of Cas9, transitioning from an inactive state (when intein is present) to an active state (when intein excises), thereby regulating the harmful off-target effects while preserving on-target activity
2Productivity
If Cas9 proteins are expressed at high levels for efficient genome editing, then on-target cleavage efficiency increases, but off-target effects and cellular toxicity worsen
Solution Approach 1:
High levels of Cas9 protein with inserted intein can be expressed and delivered into cells in advance without causing harm, because the intein keeps the nuclease activity inhibited. The high protein levels ensure sufficient on-target editing capacity is available, but the harmful off-target effects are prevented until the intein excises itself under controlled conditions
Solution Approach 2:
The invention changes the activation parameter of Cas9 from constitutive (always active) to conditional (active only when intein excises). This parameter change allows the system to maintain high protein expression levels for efficiency while controlling the actual enzymatic activity to minimize off-target effects, effectively decoupling protein quantity from harmful activity
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
The use of ligand-dependent inteins in Cas9 proteins enables precise control over enzymatic activities, reducing off-target DNA cleavage and cellular toxicity, enhancing the safety and efficacy of genomic manipulations.
Implementation Method 1
The presence of the intein inhibits one or more activities of the Cas9 proteins... The intein may be inserted into any location of a Cas9 protein... the intein replaces amino acid residue Cys80, Ala127, Thr146, Ser219, Thr333, Thr519, Cys574, Thr622, Ser701, Ala728, Thr995, Ser1006, Ser1154, Ser1159, or Ser1274
Data Source
AI summary
Some aspects of this disclosure provide compositions, methods, systems, and kits for controlling the activity of RNA-programmable endonucleases, such as Cas9, or for controlling the activity of proteins comprising a Cas9 variant fused to a functional effector domain, such as a nuclease, nickase, recombinase, deaminase, transcriptional activator, transcriptional repressor, or epigenetic modifying domain. For example, the inventive proteins provided comprise a ligand-dependent intein, the presence of which inhibits one or more activities of the protein (e.g., gRNA binding, enzymatic activity, target DNA binding). The binding of a ligand to the intein results in self-excision of the intein, restoring the activity of the protein.