CRISPR-Associated Transposase Systems for Human Genome Editing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current genome editing technologies, such as CRISPR-Cas systems, face challenges in achieving efficient and precise targeted gene modification, particularly in eukaryotic genomes, due to limitations in scalability, affordability, and ease of use.
Innovation Solution
The development of an engineered system comprising CRISPR-associated Tn7 or Tn7-like transposase polypeptides, Type I-B Cas proteins, and a guide molecule capable of directing binding to a target polynucleotide, enabling reproducible insertions with high frequency of unique insertion reads.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If CRISPR-Cas systems are used for targeted genome editing, then genome modification capability is achieved, but scalability and ease of use are limited
Solution Approach 1:
The patent introduces an intermediary mechanism by fusing Cas proteins with transposase enzymes, creating a hybrid system where the Cas protein guides the transposase to the target site. This intermediary approach allows the system to combine the targeting precision of CRISPR-Cas with the efficient DNA insertion capability of transposases, thereby improving ease of use while maintaining genome editing capability.
Solution Approach 2:
The patent merges two distinct molecular mechanisms - CRISPR-Cas guided targeting and transposase-mediated DNA insertion - into a single integrated system. By combining these functions, the system achieves both the adaptability of CRISPR for specific target recognition and the efficiency of transposases for DNA integration, resolving the contradiction between capability and ease of use.
2Adaptability or versatility
If CRISPR-Cas systems are used for targeted genome editing, then genome modification capability is achieved, but scalability is limited
Solution Approach 1:
The transposase acts as an intermediary that enhances the productivity of the CRISPR-Cas system. By recruiting the transposase to the CRISPR-Cas complex, the system leverages the transposase's efficient DNA transposition machinery to accelerate the genome editing process, thereby improving scalability while maintaining the adaptability of CRISPR for various target sites.
Solution Approach 2:
The patent creates a universal platform by designing the Cas-transposase fusion system to be applicable across multiple target sites and genome types. The modular nature of the guide RNA and the versatile transposase activity enable the system to scale to multiple editing tasks simultaneously, resolving the contradiction between genome editing capability and scalability.
3Manufacturing precision
If traditional genome editing techniques are used, then targeted perturbation is achieved, but cost and complexity increase
Solution Approach 1:
The patent extracts and utilizes only the essential functions needed for targeted perturbation - the guide RNA for target recognition and the transposase for DNA insertion - while eliminating unnecessary components of traditional systems. This extraction approach maintains high precision for targeted perturbation while reducing overall system complexity and cost.
Solution Approach 2:
The patent employs a simplified, more cost-effective system by using transposase enzymes that can be delivered as mRNA or protein, avoiding the need for complex viral vectors or prolonged expression systems. This disposable approach maintains targeted perturbation precision while reducing system complexity and cost.
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 achieves efficient and precise insertion of donor polynucleotides into target sites in eukaryotic genomes, with a frequency of unique insertion reads greater than 10%, facilitating advanced applications in genome engineering and biotechnology.
Implementation Method 1
a guide molecule capable of complexing with the Type I-B Cas protein and directing binding of the guide-Cas protein complex to a target polynucleotide
Implementation Method 2
one or more CRISPR-associated Tn7 or Tn7-like transposase polypeptides or functional fragments thereof
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 CRISPR systems and transposable elements. CRISPR-associated transposons (CASTs) are bacterial RNA-guided Tn7-like transposons that can insert large DNA cargoes at targeted loci in bacteria. To adapt CAST for use on the human genome, Applicants screened 30 CAST I-B systems for activity on extrachromosomal DNA in human cells. Of the six active orthologs identified, Applicants engineered a I-B2 system from Tolypothrix sp. PCC7910 (TolCAST) to achieve RNA-guided DNA insertion in the genome of living human cells.