Targeted Non-Viral DNA Insertion Using Cas9 RNP-DNA Complexes
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods for introducing targeted mutations or DNA sequences into cells face limitations due to size constraints of synthetic oligonucleotides and high cell death rates with larger DNA, while viral vectors cause off-target effects.
Innovation Solution
A method involving a Cas9 ribonucleoprotein (RNP)-DNA template complex is used to integrate large nucleotide sequences into a targeted genomic region, reducing off-target effects and cell viability loss by using a complex of Cas9 nuclease and guide RNA with a DNA template over 200 nucleotides, optimized through specific molar ratios and incubation conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If synthetic ssODN is used to introduce targeted DNA sequences, then sequence definition is achieved, but DNA size is limited to chemical synthesis capabilities
Solution Approach 1:
The patent segments the DNA delivery system into two functional components: (1) a Cas9-RNP complex that creates a targeted double-strand break at the genomic locus, and (2) a separate donor DNA template (ssODN or dsDNA) that provides the sequence to be inserted. This segmentation allows the donor DNA to be chemically synthesized in manageable sizes while the Cas9-RNP handles the targeted insertion function, resolving the contradiction between sequence definition and DNA size limits.
Solution Approach 2:
The patent introduces the Cas9-RNP complex as an intermediary mechanism that facilitates the integration of donor DNA into the genome. Instead of directly delivering large DNA sequences, the Cas9-RNP creates a targeted break that enables HDR of the donor template, effectively mediating the insertion process and allowing use of chemically synthesizable DNA sizes.
2Quantity of substance
If larger DNA templates are used for insertion, then insertion size increases, but cell viability decreases
Solution Approach 1:
The patent segments the insertion process into two independent steps: (1) Cas9-RNP creates a targeted double-strand break, and (2) donor DNA template is processed through HDR. This segmentation allows the cell's repair machinery to handle the insertion without being overwhelmed by large DNA templates, maintaining cell viability while enabling larger insertions than direct delivery methods.
Solution Approach 2:
The patent changes the delivery parameters by using electroporation to introduce the Cas9-RNP complex and donor DNA template separately or in controlled combinations. By optimizing the timing, concentration, and delivery method parameters, the system achieves high insertion efficiency with large DNA templates while maintaining cell viability, avoiding the toxicity associated with direct large DNA delivery.
3Quantity of substance
If viral vectors are used to deliver large DNA sequences, then insertion size increases, but off-target effects occur
Solution Approach 1:
The patent extracts the viral vector component and replaces it with a non-viral Cas9-RNP based system. The Cas9-RNP complex delivers the donor DNA template without the harmful viral elements, achieving large DNA sequence insertion while eliminating off-target effects associated with viral integration. The system uses a plasmid-based or synthetic DNA template instead of viral vectors.
Solution Approach 2:
The patent uses the Cas9-RNP complex as an intermediary that enables targeted insertion without viral vectors. The Cas9-RNP creates a controlled double-strand break at the desired locus, and the donor DNA template is then integrated through HDR, serving as a non-viral intermediary mechanism that avoids the off-target effects and safety concerns of viral vectors.
4Productivity
If NHEJ is used for mutation introduction, then insertion efficiency increases, but sequence precision is lost
Solution Approach 1:
The patent changes the repair pathway parameter by providing a donor DNA template with homology arms that promote Homology Directed Repair (HDR) instead of allowing NHEJ to dominate. By optimizing the donor template design (with flanking homology sequences) and the cellular conditions, the system shifts the repair mechanism from imprecise NHEJ to precise HDR, achieving both efficiency and sequence accuracy.
Data Source
AI summary
Provided herein are methods and compositions for editing the genome of a cell. In some embodiments, a nucleotide sequence of at least 200 nucleotides in length is inserted into a target region in the genome of a cell.