Multi-Promoter Guide RNA Cassettes for Genome Editing Efficiency
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Solution Overview
Problem
Current genome editing technologies, such as CRISPR/Cas systems, face challenges in achieving efficient site-directed integration and precise edits in eukaryotic organisms, particularly in regions where guide RNAs exhibit low cutting and editing efficiency.
Innovation Solution
The method involves expressing guide nucleic acids and guide RNAs for RNA-guided nucleases from multiple recombinant cassettes under the control of different promoters in cells, thereby increasing editing efficiency at target sites that are moderately or poorly recognized by a single guide RNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a single guide RNA is used for genome editing, then the system is simple and easy to operate, but the editing efficiency at certain target sites is low
Solution Approach 1:
The patent divides the guide RNA expression system into multiple independent recombinant cassettes, each containing a guide RNA sequence under the control of a distinct promoter. This segmentation allows simultaneous expression of multiple guide RNAs targeting the same or different genomic loci, thereby improving editing efficiency without requiring complete redesign of the entire system.
Solution Approach 2:
The patent creates a multi-functional expression system where a single construct can simultaneously express multiple guide RNAs through the use of multiple promoters. This universal approach enables the system to target multiple genomic sites or use multiple guides at a single site, enhancing versatility while maintaining a relatively streamlined architecture.
2Productivity
If multiple recombinant cassettes with different promoters are used to express guide RNAs, then editing efficiency is improved, but the device complexity increases
Solution Approach 1:
The patent segments the guide RNA expression into multiple independent cassettes, each with its own promoter, allowing for modular assembly and independent optimization of each component while achieving cumulative editing efficiency improvements.
Solution Approach 2:
The patent varies the promoter parameters (different promoter sequences, strengths, and types) to optimize guide RNA expression levels. By changing promoter characteristics rather than altering the guide RNA sequences themselves, the system achieves improved editing efficiency with minimal increase in overall complexity.
Data Source
AI summary
Compositions and methods are provided for the expression of similar guide nucleic acids for guided nucleases from multiple recombinant cassettes under control of different promoters in cells.