CRISPR-Mu Transposase Genome Editing Platform
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Solution Overview
Problem
Current genome editing technologies lack affordable, easy-to-set-up, scalable, and versatile methods for precise targeting of multiple positions within the eukaryotic genome, limiting their application in genome engineering and biotechnology.
Innovation Solution
An engineered system utilizing CRISPR-associated Mu transposases, Cas proteins, and guide molecules for sequence-specific binding and insertion of donor polynucleotides into target polynucleotides, enabling precise genome modifications such as mutations, corrections, or disruptions of splicing sites.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional genome editing technologies (designer zinc fingers, TALEs, homing meganucleases) are used, then targeted genome perturbations can be achieved, but the methods are not affordable, difficult to set up, and not scalable for multiple positions
Solution Approach 1:
The patent merges the CRISPR-Cas system (which provides sequence-specific targeting) with the Mu transposase system (which provides insertion capability) into a single integrated platform. This combination allows the system to achieve both precise targeting through guide RNA-Cas protein binding and efficient insertion through transposase activity, resolving the contradiction between targeting precision and system complexity by leveraging the strengths of two existing systems rather than developing entirely new complex tools for each function
Solution Approach 2:
The CRISPR-Cas system is adapted to serve multiple functions: it provides both the targeting function (through guide RNA-Cas protein complex formation) and the insertion function (by recruiting Mu transposase and donor DNA to the target site). This multi-functionality allows a single system to replace multiple specialized tools, improving affordability and ease of setup while maintaining targeting precision
2Manufacturing precision
If conventional genome editing technologies are used, then targeted genome perturbations can be achieved, but the methods are not scalable for targeting multiple positions within the eukaryotic genome
Solution Approach 1:
The system uses modular guide RNA molecules that can be independently designed for each target position. Multiple guide RNAs can be introduced simultaneously, each directing the Cas-Mu transposase complex to a specific genomic location. This segmentation of the targeting function into independent, interchangeable guide RNA components enables scalable targeting of multiple positions while maintaining the precision of individual targeting
Solution Approach 2:
The Mu transposase acts as an intermediary that bridges the CRISPR targeting complex and the donor DNA insertion process. It recruits the Cas-guide RNA complex to the target site and facilitates the insertion of donor polynucleotides, enabling efficient and scalable genome editing across multiple positions without requiring separate optimization for each target
3Ease of manufacture
If new genome engineering technologies are developed, then affordability and ease of setup can be improved, but targeting precision and versatility may be compromised
Solution Approach 1:
The system uses guide RNA molecules that are synthesized in vitro and introduced into cells, rather than requiring complex in vivo assembly or expression from large plasmid constructs. This copying approach simplifies the setup process while maintaining targeting precision, as the guide RNAs can be designed and synthesized with high accuracy and introduced directly into the cell 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 system allows for efficient and targeted genome editing, enhancing the capability for systematic reverse engineering of genetic variations and advancing synthetic biology and medical applications by providing a versatile and scalable method for genome engineering.
Implementation Method 1
a guide molecule capable of complexing with the Cas protein and directing sequence-specific binding of the guide-Cas protein complex to the target polynucleotide
Implementation Method 2
one or more CRISPR-associated Mu transposases; one or more Cas proteins; and a guide molecule capable of complexing with the Cas protein and directing sequence-specific binding of the guide-Cas protein complex to the target polynucleotide
Data Source
AI summary
Systems and methods for targeted gene modification, targeted insertion, perturbation of gene transcripts, and nucleic acid editing. Novel nucleic acid targeting systems comprise components of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems and transposable elements.