Fusion Protein HDR System for Precise Gene Editing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current CRISPR/Cas9 genome editing techniques face limitations in efficiency and specificity for homology-directed repair (HDR), particularly due to high frequencies of frameshift mutations and low integration rates of genetic material, which hinder precise gene editing for various diseases and disorders.
Innovation Solution
A method involving the delivery of a gene editing system comprising guide RNAs and fusion proteins with RNA programmable nuclease and exonuclease domains to create double-strand breaks between specific genomic sites, promoting HDR and insertion of a donor DNA molecule, thereby enhancing gene editing precision and efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If CRISPR/Cas9 is used for genome modification, then both introduction and deletion of genetic material are enabled, but the efficiency of homology directed repair is low
Solution Approach 1:
The Cas9 nuclease is divided into two separate components: a dead Cas9 (dCas9) that binds to the target site without cleaving, and a separate active Cas9 that creates the double-strand break. This segmentation allows independent optimization of each component's function, enabling precise control over where the break occurs and how the repair process is initiated.
Solution Approach 2:
A single-stranded DNA oligonucleotide donor is introduced as an intermediary molecule that bridges the double-strand break and the desired genetic modification. This donor template serves as the blueprint for homology-directed repair, guiding the cellular repair machinery to integrate the precise genetic sequence desired by the researcher.
2Productivity
If non-homologous end joining is used for repair, then frameshift mutations occur at high efficiency, but precise genetic material integration is lost
Solution Approach 1:
The system dynamically switches between two repair pathways by controlling the timing and availability of repair templates. When a single-stranded DNA donor is provided, the system favors homology-directed repair; when no template is available, non-homologous end joining occurs naturally. This dynamic control allows selection of the appropriate repair mechanism based on experimental needs.
Solution Approach 2:
The invention changes the physical state of the DNA donor from double-stranded to single-stranded form. Single-stranded DNA donors are more efficiently incorporated into the HDR pathway compared to double-stranded DNA, as they more closely resemble the natural repair template and require less processing by cellular enzymes.
3Manufacturing precision
If homology directed repair is used for genetic material integration, then precise knock in is achieved, but the efficiency is low
Solution Approach 1:
The invention changes the physical state of the DNA donor from double-stranded to single-stranded form. Single-stranded DNA donors are more efficiently incorporated into the HDR pathway compared to double-stranded DNA, as they more closely resemble the natural repair template and require less processing by cellular enzymes. This parameter change increases HDR efficiency while maintaining precision.
Solution Approach 2:
The system performs preliminary action by creating a clean double-strand break with precise boundaries before the repair process begins. The Cas9 nuclease cuts at a defined location, creating uniform ends that are primed for efficient HDR. This preliminary precision cutting prepares the site optimally for subsequent high-efficiency template integration.
4Productivity
If multiple nucleases are used to create breaks between genomic sites, then HDR efficiency increases, but the complexity of the gene editing system increases
Solution Approach 1:
Multiple guide RNAs that would normally require separate delivery and coordination are merged into a single polycistronic transcript. This single RNA molecule contains multiple guide sequences that are processed individually by the cell's RNA processing machinery, allowing multiple target sites to be addressed simultaneously while simplifying the delivery system to a single RNA component.
Solution Approach 2:
The Cas9 nuclease is designed with universal functionality to recognize and bind any guide RNA sequence, allowing a single Cas9 protein to mediate cleavage at multiple different genomic locations. This multi-functionality eliminates the need for multiple different nuclease proteins, reducing system complexity while enabling efficient HDR at multiple sites.
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 significantly increases the efficiency and specificity of HDR, allowing for precise correction or introduction of genetic material at targeted sites, potentially treating a wide range of diseases and disorders by promoting accurate homology-directed repair mechanisms.
Implementation Method 1
a first guide ribonucleic acid (RNA) directed to a first genomic site of an endogenous DNA molecule of the target cell, ii) a second guide RNA directed to a second genomic site of the endogenous DNA molecule of the target cell, iii) a plurality of fusion proteins comprising a first domain comprising an active RNA programmable nuclease
Implementation Method 2
a second domain comprising an exonuclease, and, optionally, and iv) a donor DNA molecule, in which the first guide RNA forms a first complex with a first said fusion protein at the first genomic site and the second guide RNA forms a second complex with a second said fusion protein at the second genomic site
Implementation Method 3
the first and second complexes promote the homology directed repair by creating a lesion (e.g., double strand break) between the first and second genomic sites and, optionally, where the homology directed repair comprises insertion of the donor DNA molecule at the lesion between the first and second genomic sites
Data Source
AI summary
Described are methods of homology directed repair (e.g., for treating a disease or disorder) and fusion proteins, polynucleotides (e.g., guide polynucleotides (e.g., guide RNAs) and polynucleotides encoding the fusion proteins), vectors containing the polynucleotides, viral or non-viral delivery vehicles containing the vectors, and compositions (e.g., pharmaceutical compositions) containing the same for use in methods of homology directed repair.