ELSA sgRNA Arrays for Stable Multi-Target CRISPR Editing
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Solution Overview
Problem
Existing CRISPR-based systems face challenges in simultaneously and stably expressing multiple guide RNAs due to repetitive DNA sequences, which lead to genetic instability and spontaneous deletions, particularly in microbial organisms and mammalian genetic engineering, hindering broader applications in biotechnology.
Innovation Solution
The development of Extra Long Single-Guide RNA Arrays (ELSAs) with non-repetitive sgRNA promoters, handles, and spacers, allowing for the simultaneous expression of multiple sgRNAs with minimal recombination, using a maximum shared repeat length of 20 nucleotides or less, and incorporating RNA-guided enzymes like Cas9 for targeted gene modulation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple guide RNAs are co-expressed using traditional sgRNA arrays with repetitive DNA sequences, then the ability to simultaneously target multiple genomic locations is improved, but genetic stability deteriorates due to homologous recombination and spontaneous deletions
Solution Approach 1:
The patent segments the sgRNA array into multiple individual expression units, each containing a unique guide RNA sequence flanked by non-repetitive promoter and terminator sequences. This segmentation eliminates long repetitive DNA sequences that trigger homologous recombination, allowing simultaneous expression of multiple sgRNAs while maintaining genetic stability.
Solution Approach 2:
The patent applies local quality by designing each sgRNA expression unit with site-specific non-repetitive sequences tailored to that particular guide RNA. Each unit has its own unique promoter and terminator sequences, preventing homologous recombination between adjacent units while maintaining local transcriptional control for optimized sgRNA expression at each target location.
2Device complexity
If repetitive DNA sequences are used in sgRNA arrays to enable co-expression of multiple guide RNAs, then the complexity of targeting multiple locations is reduced, but the system reliability deteriorates due to spontaneous deletions
Solution Approach 1:
The sgRNA array is segmented into discrete expression cassettes, each with unique non-repetitive boundary sequences. This segmentation maintains relatively simple device architecture for multi-targeting while eliminating the harmful repetitive sequences that cause spontaneous deletions and reduce system reliability.
Solution Approach 2:
The patent introduces non-repetitive promoter and terminator sequences as intermediary elements between adjacent sgRNA units. These intermediary sequences act as buffers that prevent homologous recombination while allowing each sgRNA to be independently expressed, thus maintaining system reliability without compromising the ability to target multiple locations.
3Ease of manufacture
If traditional sgRNA arrays with long repetitive sequences are used, then assembly in vitro becomes more difficult, but the patent improves ease of manufacture by using non-repetitive sequences
Solution Approach 1:
The sgRNA array is designed as segmented modular units with non-repetitive sequences, which significantly improves ease of in vitro assembly by eliminating complex homologous recombination events. Each module can be independently synthesized and assembled, greatly simplifying the manufacturing process while simultaneously improving genetic stability in vivo.
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
ELSAs enable stable and simultaneous modulation of multiple genes, reducing genetic instability and enhancing the application of CRISPR technology in biotechnology by allowing for precise regulation of gene expression without introducing repetitive DNA sequences, thereby improving the treatment of genetic diseases and studying complex gene networks.
Implementation Method 1
Engineered CRISPR-based systems have been applied to bind, edit, and cut genomic DNA at specified locations
Data Source
AI summary
The invention provides CRISPR-based compositions and methods comprising non-repetitive sgRNA promoter and handle sequences for simultaneous, stable expression of multiple sgRNAs.