rAAV CasX Vectors for Size-Limited Targeted Gene Editing
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Solution Overview
Problem
Safe and targeted delivery of CRISPR gene editing machinery to desired cells remains a challenge for effective therapeutic applications.
Innovation Solution
Recombinant adeno-associated virus (rAAV) vectors are developed to deliver Class 2, Type V CRISPR proteins and guide nucleic acids, incorporating CasX nucleases and gRNA, with optimized sequences to minimize immunogenicity and accommodate complete nuclease and multiple gRNA components, enabling efficient transduction and expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If Cas9 nuclease and multiple gRNA components are included in the vector, then complete gene editing functionality is achieved, but the vector size exceeds the packaging capacity of rAAV
Solution Approach 1:
The patent extracts and replaces the Cas9 nuclease with the smaller CasX nuclease (Type V CRISPR protein), which has similar gene editing functionality but a smaller molecular size. This extraction of the essential function (nuclease activity) while removing the bulky Cas9 component allows the transgene to fit within the rAAV packaging capacity of approximately 4.7-5 kb, enabling delivery of complete nuclease and multiple gRNA components.
Solution Approach 2:
The patent changes the key parameter of nuclease size by switching from Cas9 to CasX. The CasX nuclease has a smaller molecular weight and coding sequence length compared to Cas9, which directly reduces the transgene size while maintaining the essential gene editing function. This parameter change enables the vector to accommodate complete nuclease and multiple gRNA components within the limited packaging capacity.
2Productivity
If AAV vector components are used for gene delivery, then efficient transduction is achieved, but immunogenicity is triggered by CpG dinucleotides in the vector sequences
Solution Approach 1:
The patent identifies CpG dinucleotides in AAV vector components as triggers for immune responses. By systematically removing or mutating these CpG dinucleotides from the vector sequences (including ITRs, promoters, encoding sequences, and poly(A) signals), the patent converts a harmful immunogenic feature into a beneficial non-immunogenic vector while preserving the essential transduction functionality. This creates an immunologically optimized AAV vector system.
3Volume of moving object
If transgene size is reduced to fit rAAV packaging capacity, then functional rAAV particles are formed, but complete CRISPR components and accessories cannot be included
Solution Approach 1:
The patent segments the CRISPR system into essential and non-essential components. By using the smaller CasX nuclease, the patent creates space to include multiple gRNA components and necessary accessory elements (promoters, poly(A) signals, ITRs) within the rAAV packaging capacity. The segmentation allows prioritization of critical gene editing components while maintaining functional completeness.
Solution Approach 2:
The patent designs a universal rAAV vector system that can accommodate complete CRISPR components (nuclease and multiple gRNAs) plus accessory elements within the limited packaging capacity. The use of compact CasX enables this multi-functional vector to deliver a complete gene editing toolkit, making the system universally applicable for various gene editing applications.
Data Source
AI summary
Provided herein are recombinant adeno-associated virus (rAAV) compositions and methods for use of the rAAV encoding CasX proteins and guide ribonucleic acid (gRNA) sequences useful for nucleic acid sequence editing, and including transgene components. The rAAV may be delivered to cells to target a gene of interest.