Engineered gRNA Scaffolds for Higher Type V CRISPR Editing
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Solution Overview
Problem
Existing Class 2, Type V CRISPR/Cas systems exhibit low editing efficiency and require improvements for therapeutic, diagnostic, and research applications.
Innovation Solution
Development of engineered Class 2, Type V CRISPR proteins and guide ribonucleic acids (gRNAs) with modified domains, such as extended stem loops, scaffold stem loops, triplexes, and pseudoknots, to enhance binding and cleavage efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type Class 2, Type V CRISPR/Cas systems are used, then the system structure is simple and easy to implement, but the editing efficiency is low
Solution Approach 1:
The patent applies parameter changes by modifying the guide RNA scaffold structure through engineered mutations. Specific changes include altering the extended stem loop region, scaffold stem loop, triplex structure, and pseudoknot formation. These structural parameter modifications to the gRNA scaffold enhance the binding affinity and stability of the CasX-gRNA complex, thereby improving editing efficiency while maintaining the fundamental simplicity of the Class 2, Type V system architecture
Solution Approach 2:
The patent employs composite material principles by creating engineered hybrid gRNA scaffolds that combine multiple structural elements with optimized properties. The modified scaffold integrates extended stem loops with specific secondary structures, triplex regions, and pseudoknot formations that work synergistically. This composite structural design enhances the overall functionality and efficiency of the CRISPR system while preserving its core simplicity
2Reliability
If the guide RNA scaffold structure is modified to improve binding efficiency, then the binding affinity increases, but the design and manufacturing complexity increases
Solution Approach 1:
The patent applies segmentation by dividing the gRNA scaffold into distinct functional modules that can be independently optimized and assembled. The scaffold is segmented into the extended stem loop region, scaffold stem loop, triplex structure, and pseudoknot regions. Each segment can be independently designed and modified to achieve optimal binding affinity, while the modular nature simplifies the overall design process and facilitates manufacturing by allowing standardized assembly of proven functional elements
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 engineered systems demonstrate improved nucleic acid editing capabilities, including increased specificity and efficiency, suitable for gene editing in eukaryotic cells.
Implementation Method 1
the gRNA comprises at least one modification in a region compared to the gRNA of SEQ ID NO: 2238 or SEQ ID NO: 2239... capable of binding a Class 2, Type V variant protein
Implementation Method 2
the complex can bind and cleave a target nucleic acid, wherein the target nucleic acid comprises a non-target strand and a target strand
Data Source
AI summary
Provided herein are engineered Class 2. Type V nucleases and guide RNAs useful for the editing of target nucleic acids. Also provided are methods of making and using such variants to modify nucleic acids.