Split SaCas9 CRISPR System for AAV Delivery
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Solution Overview
Problem
The CRISPR/Cas system's therapeutic applications are limited by the restrictive cargo size of existing viral delivery vehicles, which restricts the integration of CRISPR/Cas components into mammalian cells, and current delivery systems often induce an immune response and have limited capacity for synthetic biology programming.
Innovation Solution
The CRISPR/Cas system is split into smaller regions and delivered using separate viral vectors, allowing for reconstitution in cells, and utilizing downsized Cas9 variants and optimized gRNA expression to accommodate additional genetic elements, enabling efficient genetic manipulation and reduced immune response.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the entire Cas9 complex is delivered using a single viral delivery system, then complete genetic manipulation functionality is achieved, but the cargo size exceeds the packaging capacity of viral vectors
Solution Approach 1:
The Cas9 complex is divided into multiple separate components (e.g., Cas9 protein, gRNA, and optional effector domains) that are delivered using separate viral delivery vectors. This segmentation allows each component to be packaged within the 4.7-5 kb capacity limit of AAV vectors while maintaining the complete functional system upon co-delivery and assembly in target cells.
2Quantity of substance
If traditional viral delivery systems are used, then CRISPR/Cas components can be delivered, but the cargo size is restricted to 4.7-5 kb which is insufficient for complete Cas9 complex
Solution Approach 1:
The delivery system is segmented into multiple viral vectors, each carrying a specific component of the CRISPR/Cas system. This allows the total cargo capacity to exceed the limitation of individual vectors while ensuring complete delivery of all necessary elements for functional reconstitution.
Solution Approach 2:
Multiple viral vectors are used in a nested delivery approach where each vector encapsulates a specific component, and collectively they deliver the complete system. The vectors work in concert to achieve complete delivery despite individual size constraints.
3Adaptability or versatility
If standard Cas9 is used in viral delivery, then full genetic editing capability is achieved, but the size (over 4.2 kb with promoter) exceeds AAV packaging capacity (4.7-5 kb)
Solution Approach 1:
The promoter sequences and other regulatory elements are extracted and placed in a separate viral vector from the Cas9 coding sequence. This allows the Cas9 open reading frame to be packaged in one vector while the promoter and other elements are delivered in additional vectors, enabling complete functionality within AAV packaging constraints.
Solution Approach 2:
The Cas9 expression system is segmented into multiple components (promoter, Cas9 coding sequence, gRNA, etc.) distributed across multiple viral vectors. This segmentation enables each vector to remain within size limits while the collective system provides full genetic editing capability.
4Productivity
If complete CRISPR/Cas system is packaged in single vector, then all components are delivered together, but immune response is triggered and packaging capacity is wasted
Solution Approach 1:
The CRISPR/Cas system components are segmented and delivered separately using multiple viral vectors. This reduces the immunogenicity of each individual vector payload compared to a single large vector, and allows for staggered delivery that can reduce peak immune activation.
Solution Approach 2:
Separate viral vectors act as intermediaries that deliver individual components rather than the complete system in one package. This intermediary approach reduces the recognition of the complete system as a foreign entity, thereby reducing immune response.
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 enhances the capacity for genetic editing and regulation in mammalian cells, overcoming size limitations and immune response issues, facilitating more effective synthetic biology applications and therapeutic interventions.
Implementation Method 1
With intein-mediated split Cas9, inteins function as protein introns and are excised out of a sequence and join the remaining flaking regions (exteins) with a peptide bond without leaving a scar.
Data Source
AI summary
The presently claimed invention offers programmable and precise regulation of Cas9 functions by utilizing a set of compact Cas9 derivatives created by deleting conserved HNH and/or REC-C domains based on the structural information across variant class 2 CRISPR effectors. In addition, a novel strategy for engineering the dimeric gRNA-guided nuclease by splitting the mini-dSaCas9 and fusing the FokI domain right after the split point is claimed to increase the on-target DNA cleavage efficiency and potentially reduce the off-target effect because of a closer proximity of dimeric Fold nuclease to the target sequence. By combining the optimized and compact gRNA expression cassette and the downsized SaCas9 derivatives, the entire CRISPR/Cas system with different effector domains for transactivation, DNA cleavage and base editing is loaded into a single AAV virus. Such an all-in-one AAV-CRISPR/Cas9 system will be particularly appealing in biomedical applications that require safe and efficient delivery in vivo.