Cas12f1 Gene Editing System for AAV Delivery of USH2A Exon 13
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Solution Overview
Problem
Current CRISPR/Cas systems have low intracellular gene editing activity and difficulty in vivo delivery due to their large molecular weight, limiting their therapeutic effectiveness for treating Usher syndrome, particularly in delivering exon 13 deletion for USH2A gene mutations.
Innovation Solution
A CRISPR/Cas12f1 system with engineered guide RNAs and a hypercompact Cas12f1 protein is developed to specifically target and delete exon 13 of the USH2A gene, utilizing a smaller endonuclease and optimized guide RNAs for efficient gene editing, compatible with delivery vehicles like adeno-associated virus (AAV).
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a conventional CRISPR/Cas system is used for gene editing, then gene editing capability is achieved, but the molecular weight is too large for efficient in vivo delivery
Solution Approach 1:
The patent segments the CRISPR system into a smaller Cas12f1 endonuclease component and separate guide RNA components. This segmentation allows the endonuclease to be small enough for AAV delivery while maintaining gene editing functionality, resolving the contradiction between editing capability and deliverability.
Solution Approach 2:
The patent extracts and utilizes only the essential endonuclease activity from the CRISPR system by employing Cas12f1, a minimal endonuclease from Class 2 Type F CRISPR systems. This extraction of core functionality enables delivery within size-constrained vectors while preserving gene editing capability.
2Productivity
If the CRISPR system is miniaturized for delivery, then delivery efficiency improves, but gene editing activity decreases
Solution Approach 1:
The patent changes the parameters of the CRISPR system by selecting Cas12f1, which has different physical and functional characteristics compared to larger Cas9 systems. This parameter change (using a smaller endonuclease with specific PAM requirements) enables both efficient delivery and maintained editing activity.
Solution Approach 2:
The patent optimizes the guide RNA sequences specifically for Cas12f1 recognition and binding, creating locally optimized components that maximize editing efficiency within the constrained system. This local optimization ensures that despite the miniaturized system, gene editing activity remains high.
3Reliability
If exon 13 deletion is achieved in USH2A gene, then therapeutic effect improves, but delivery to target tissue becomes more difficult
Solution Approach 1:
The patent uses adeno-associated virus (AAV) as an intermediary delivery vehicle to transport the miniaturized CRISPR/Cas12f1 system to target tissues. This intermediary enables in vivo delivery of the gene editing components, making the therapeutic approach feasible while achieving the desired exon 13 deletion.
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 system achieves enhanced gene editing efficiency and specificity, allowing for effective treatment of Usher syndrome by inducing exon 13 deletion, producing functional Usherin protein, and is suitable for use in various delivery vehicles, overcoming size limitations of previous systems.
Implementation Method 1
a first guide RNA comprising a first guide sequence capable of hybridizing to a target sequence of contiguous 15 to 30 bp in length, wherein the target sequence is located in a region 5000 bp upstream of USH2A exon 13
Implementation Method 2
an endonuclease comprising a Cas12f1 molecule... adjacent to a protospacer-adjacent motif (PAM) sequence recognized by the Cas12f1 molecule
Data Source
AI summary
The present invention relates to a gene editing system for treating Usher syndrome, to a disease treatment method using same, and the like, wherein by using the gene editing system of the present invention, an exon 13 region mutated in the Usherin (USH2A) gene is deleted with high efficiency, thereby treating type 2 Usher syndrome effectively. In addition, the gene editing system of the present invention comprises a miniaturized Cas12f1 protein-based endonuclease and an engineered guide RNA having a shorter length and improved indel efficiency, and a carrier having a limited packaging size, such as adeno-associated virus (AAV), can also be used, and therefore, in vivo or intracellular delivery efficiency can also be maximized.