Dual Guide CRISPR RNA Architecture for Specific Genome Editing
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Solution Overview
Problem
Existing CRISPR-Cas systems lack sufficient flexibility and specificity for precise genome editing, particularly in applications involving genetically engineered cells for therapy, leading to off-target editing issues.
Innovation Solution
A dual guide CRISPR-Cas system is engineered, comprising a targeter and modulator nucleic acid that activate a Cas nuclease, allowing for adjustable hybridization length and affinity to enhance specificity and flexibility, reducing off-target editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If a single guide RNA is used to activate Cas nuclease, then the system is simple and easy to operate, but the specificity and flexibility for precise genome editing are insufficient
Solution Approach 1:
The patent divides the single guide RNA into two separate nucleic acid components: a targeter nucleic acid (containing the spacer sequence) and a modulator nucleic acid (containing the modulator sequence). This segmentation allows each component to have optimized functions - the targeter for target recognition and the modulator for regulating Cas nuclease activation - thereby improving editing specificity while maintaining operational simplicity through modular design
2Manufacturing precision
If the guide RNA is extended to improve specificity, then the specificity increases, but the synthesis yield and accuracy decrease due to length limitations
Solution Approach 1:
By splitting the guide RNA into two shorter separate nucleic acid sequences (targeter and modulator), each component can be synthesized with high yield and accuracy while collectively providing the enhanced specificity needed for precise genome editing. The modular structure avoids the synthesis problems associated with long single-stranded RNAs
3Adaptability or versatility
If a dual guide system with adjustable hybridization is implemented, then the flexibility and specificity are enhanced, but the device complexity increases
Solution Approach 1:
The system is divided into modular components (targeter nucleic acid with spacer, modulator nucleic acid with modulator sequence) that can be independently designed and optimized. This segmentation provides flexibility and adaptability for different editing applications while maintaining manageable complexity through standardized modular elements
Solution Approach 2:
The dual guide system creates a universal platform that can be adapted for various genome editing applications by adjusting the hybridization parameters between targeter and modulator sequences. The same basic architecture serves multiple functions including regulating Cas nuclease activation, controlling editing efficiency, and enabling flexible target selection
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 dual guide system achieves high specificity and efficiency in nucleic acid editing, particularly in mammalian cells, including stem cells and immune memory cells, by minimizing off-target effects.
Implementation Method 1
a targeter nucleic acid and a modulator nucleic acid, when hybridized to form a complex, can activate a Cas nuclease
Data Source
AI summary
The present invention relates to an engineered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system comprising engineered dual guide nucleic acids (e.g., RNAs) capable of activating a CRISPR-Associated (Cas) nuclease, such as a type V-A Cas nuclease. Also provided are methods of targeting, editing, and/or modifying a nucleic acid using the engineered CRISPR system, and compositions and cells comprising the engineered CRISPR system.