Multiplex CRISPR Array for Simultaneous Gene Up- and Downregulation
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Solution Overview
Problem
Current methods are unable to simultaneously and efficiently regulate multiple genes in cells, limiting their application in cell reprogramming and therapy due to the inability to upregulate and downregulate a large number of genes simultaneously.
Innovation Solution
Engineered multiplex CRISPR arrays comprising multiple CRISPR RNAs with specific repeat sequences and spacers, including Cas12a and Cas13, enable simultaneous upregulation and downregulation of multiple target genes using a single construct, incorporating a short AT-rich separator sequence to improve performance.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple separate expression vectors are used to regulate multiple genes, then each gene can be regulated individually, but the device complexity and labor intensity increase significantly
Solution Approach 1:
The patent combines multiple crRNA expression cassettes into a single CRISPR array construct that can simultaneously target multiple genes. This single construct integrates the functionality of multiple separate plasmids, reducing device complexity while maintaining the ability to regulate multiple genes through coordinated crRNA expression from the array
Solution Approach 2:
The CRISPR array construct serves multiple functions simultaneously: it can regulate both upregulation and downregulation of multiple genes through different crRNAs, replace multiple separate expression vectors, and provide coordinated gene regulation from a single integrated platform, thereby achieving multi-functionality that reduces the number of components needed
2Productivity
If traditional CRISPR methods are used to regulate a few genes, then the system is simple to design, but the productivity and throughput are limited
Solution Approach 1:
The CRISPR array is segmented into multiple crRNA expression cassettes, each targeting a specific gene. This segmentation allows the system to simultaneously regulate multiple genes (increasing productivity) while maintaining the modular structure that facilitates efficient design and implementation, thereby reducing the time required for complex multi-gene reprogramming operations
3Manufacturing precision
If multiple separate gene editing tools are used for upregulation and downregulation, then each gene can be precisely controlled, but the ease of operation and scalability decrease
Solution Approach 1:
The patent merges upregulation and downregulation capabilities into a single CRISPR array construct with multiple crRNA cassettes. This unified approach maintains precise control over individual gene expression while dramatically improving ease of operation and scalability, as the single construct can be delivered and implemented systemically without requiring coordination of multiple separate tools
Solution Approach 2:
The CRISPR array provides universal functionality for both upregulation and downregulation of multiple genes through a single platform. This multi-functional design maintains the precision of individual gene control while enabling scalable application to large-scale cell engineering and therapy through a unified, easily operable system
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
This approach allows for precise control of gene expression in cells, enabling efficient regulation of multiple genes simultaneously, enhancing the capabilities of CRISPR systems for cell engineering and therapy.
Implementation Method 1
the spacer is configured to hybridize to a specific target nucleic acid of a plurality of target nucleic acids
Data Source
AI summary
The present disclosure generally relates to compositions and methods simultaneous, multi-mode gene expression regulation (e.g., simultaneous upregulation and down regulation of multiple target genes). The present disclosure further relates to novel constructs for engineered multiplex CRISPR arrays.