Switchable Cas9 gRNA Control for Off-Target Cleavage Reduction
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Solution Overview
Problem
Existing RNA-guided nucleases 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 switchable guide RNAs (gRNAs) comprising aptamers that only bind and mediate DNA cleavage in the presence of a specific ligand, allowing controlled nuclease activity through conformational changes induced by ligand binding.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If RNA-guided nucleases are used for targeted genome manipulation, then site-specific cleavage capability is improved, but off-target activity increases leading to cellular toxicity
Solution Approach 1:
The gRNA is designed with a switchable aptamer domain that can dynamically change conformation based on ligand binding. In the absence of ligand, the aptamer adopts a conformation that prevents Cas9 binding. Upon ligand binding, the aptamer undergoes conformational change to allow Cas9 binding and target cleavage. This dynamic switching mechanism enables temporal control of nuclease activity, reducing off-target effects while maintaining site-specific cleavage capability when activated.
Solution Approach 2:
A small molecule ligand serves as an intermediary to control the activity of the RNA-guided nuclease complex. The ligand binds to the aptamer domain of the gRNA, inducing conformational changes that activate or inhibit Cas9 binding and DNA cleavage activity. This intermediary mechanism provides an external control knob for regulating nuclease activity, thereby reducing harmful off-target effects.
2Productivity
If nuclease activity is continuously active, then productivity of genome manipulation is improved, but off-target effects increase
Solution Approach 1:
The nuclease activity is made periodic rather than continuous through ligand-induced switching. The system remains inactive (aptamer bound to gRNA, preventing Cas9 binding) until ligand is introduced, at which point activity is activated. This periodic activation allows the system to maintain high productivity during active phases while minimizing off-target effects during inactive phases, effectively decoupling continuous operation from harmful effects.
Solution Approach 2:
The invention ensures continuity of useful action by maintaining the nuclease complex in an inactive but ready state. The aptamer-gRNA-Cas9 complex remains assembled and capable of immediate activation upon ligand binding, ensuring that when activation is needed, the system is already prepared to perform site-specific cleavage without delay, thus maintaining productivity continuity while avoiding unnecessary off-target activity during inactive periods.
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
Enhances specificity and reduces off-target effects by enabling precise control over nuclease activity, making it suitable for clinical and research applications.
Implementation Method 1
allowing controlled nuclease activity through conformational changes induced by ligand binding
Data Source
AI summary
Some aspects of this disclosure provide compositions, methods, systems, and kits for controlling the activity and/or improving the specificity of RNA-programmable endonucleases, such as Cas9. For example, provided are guide RNAs (gRNAs) that are engineered to exist in an “on” or “off” state, which control the binding and hence cleavage activity of RNA-programmable endonucleases. Some aspects of this disclosure provide mRNA-sensing gRNAs that modulate the activity of RNA-programmable endonucleases based on the presence or absence of a target mRNA. Some aspects of this disclosure provide gRNAs that modulate the activity of an RNA-programmable endonuclease based on the presence or absence of an extended DNA (xDNA).