Dead Guide RNA for CRISPR Transcription Control
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Solution Overview
Problem
Current genome editing technologies, such as CRISPR-Cas9, face challenges in achieving efficient and specific targeting of multiple positions within the eukaryotic genome while minimizing off-target effects and being affordable, easy to set up, and scalable.
Innovation Solution
The development of a non-naturally occurring CRISPR-Cas9 system comprising a functional Cas9 protein and a single guide RNA (sgRNA) with a dead guide sequence, which hybridizes to a target sequence without inducing detectable indel activity, allowing for precise gene editing without nuclease activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If CRISPR-Cas9 system uses wild-type Cas9 nuclease for genome editing, then genome editing efficiency is improved, but off-target effects and indel activity increase reducing precision
Solution Approach 1:
The guide RNA is segmented into two functional parts: a dead guide sequence (complementarity to target) and a blocking sequence (overlapping with dead guide). This segmentation allows the dead guide to bind target DNA while the blocking sequence prevents Cas9 nuclease activation, resolving the contradiction between binding efficiency and nuclease activation precision
Solution Approach 2:
The blocking sequence acts as an intermediary element that interferes with the interaction between the dead guide sequence and the Cas9 nuclease active site. This intermediary prevents harmful nuclease activity while allowing the dead guide to maintain its target-binding function, thus improving precision without sacrificing efficiency
2Manufacturing precision
If CRISPR-Cas9 system is designed for high specificity targeting, then off-target effects are reduced, but targeting efficiency and scalability decrease
Solution Approach 1:
The blocking sequence serves multiple functions simultaneously: it blocks Cas9 nuclease activity, maintains guide RNA stability, and enables scalable application across multiple target sites. This multi-functionality allows high specificity targeting to be achieved without compromising efficiency or scalability
Solution Approach 2:
The invention changes the parameters of the guide RNA by incorporating a blocking sequence with specific length and complementarity characteristics. This parameter modification allows the system to maintain high targeting specificity while preserving efficiency, as the blocking sequence can be optimized for different target contexts
3Manufacturing precision
If custom proteins are generated for each target sequence, then targeting specificity is improved, but device complexity and cost increase
Solution Approach 1:
Instead of creating custom proteins for each target, the invention uses a standardized Cas9 nuclease paired with customized guide RNAs containing dead guide and blocking sequences. This copying approach allows the same Cas9 protein to be reused across multiple targets by simply changing the RNA guide, dramatically reducing system complexity while maintaining targeting specificity
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 enables efficient and specific gene editing with reduced off-target effects, enhancing the precision and safety of genome engineering applications.
Implementation Method 1
the sgRNA is capable of hybridizing to a target sequence
Data Source
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AI summary
The invention provides for systems, methods, and compositions for altering expression of target gene sequences and related gene products. Provided are structural information on the Cas protein of the CRISPR-Cas system, use of this information in generating modified components of the CRISPR complex, vectors and vector systems which encode one or more components or modified components of a CRISPR complex, as well as methods for the design and use of such vectors and components. Also provided are methods of directing CRISPR complex formation in eukaryotic cells and methods for utilizing the CRISPR-Cas system. In particular the present invention comprehends optimized functional CRISPR-Cas enzyme systems.