Recombinant Fusion Protein for Efficient Genomic Editing
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Solution Overview
Problem
Current genome editing technologies, such as CRISPR-Cas, face challenges in efficiently integrating large donor DNA sequences into the genome due to stochastic processes and spatial restrictions, leading to low integration efficiency and cytotoxicity from high linear DNA concentrations.
Innovation Solution
A recombinant fusion protein is developed, combining a CRISPR endonuclease with a DNA binding moiety like CCR5 zinc finger, which recruits linear DNA inserts closer to the genomic cleavage site, enhancing integration efficiency and allowing lower DNA concentrations without cytotoxicity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If high linear DNA concentrations are used to improve integration efficiency, then integration efficiency increases, but cytotoxicity increases
Solution Approach 1:
The patent introduces a DNA-binding domain as an intermediary component that specifically binds to the donor DNA and recruits it to the cleavage site. This mediator enables efficient integration at lower DNA concentrations by facilitating localized accumulation of donor DNA at the target site, thereby reducing cytotoxicity while maintaining integration efficiency.
Solution Approach 2:
The fusion protein creates a localized concentration of donor DNA at the cleavage site through the DNA-binding domain. Instead of relying on high global DNA concentration, the system achieves high local concentration where needed, improving integration efficiency without the systemic cytotoxic effects of high DNA concentrations.
2Manufacturing precision
If long homology arms are used to improve knock-in efficiency, then integration accuracy improves, but integration efficiency decreases due to stochastic processes
Solution Approach 1:
The DNA-binding domain performs preliminary action by pre-recruiting and positioning the donor DNA at the cleavage site before the homologous recombination process begins. This pre-positioning overcomes the stochastic limitations of long-range diffusion and increases the probability of successful integration, particularly for large DNA fragments.
Solution Approach 2:
The DNA-binding domain acts as a mediator that bridges the donor DNA and the cleavage site, facilitating their association. This intermediary function accelerates the integration process and improves efficiency while maintaining the accuracy provided by the homology arms.
3Productivity
If short homology arms are used to improve integration speed, then integration efficiency improves, but integration accuracy decreases due to reliance on emergency DNA repair systems
Solution Approach 1:
The fusion protein creates a localized environment at the cleavage site that favors accurate homologous recombination. By concentrating the donor DNA and repair machinery at the target site, the system achieves high integration efficiency with short homology arms while maintaining accuracy through the controlled local conditions rather than relying on error-prone emergency repair systems.
4Adaptability or versatility
If large donor DNA fragments are used to improve knock-in functionality, then functional completeness improves, but integration efficiency decreases due to spatial restrictions
Solution Approach 1:
The DNA-binding domain serves as an essential intermediary for large DNA fragments. It specifically recognizes and binds to the donor DNA, bringing it into close proximity with the cleavage site. This mediated interaction overcomes the spatial restrictions that would otherwise prevent efficient integration of large fragments, enabling functional completeness without sacrificing integration efficiency.
Solution Approach 2:
The invention changes the key parameter of donor DNA concentration from a global parameter to a localized parameter at the cleavage site. By using the DNA-binding domain to create high local concentration, the system can efficiently integrate large fragments that would be impossible to integrate at physiologically relevant global concentrations.
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 integration efficiency of large DNA fragments into the genome, enabling more precise and efficient genomic editing with reduced cytotoxicity, as demonstrated by improved GFP integration in beta-actin reporter systems.
Implementation Method 1
a DNA binding moiety which binds to the linear DNA insert
Implementation Method 2
a CRISPR endonuclease with a guide RNA that directs the endonuclease to a target sequence in the genome
Data Source
AI summary
Described herein are methods and compositions for genomic editing. Endonucleases for genomic editing involve inducing breaks in double stranded DNA, for which knock-ins are notoriously inefficient for relying on random integration of homologous DNA sequences into the break site by repair proteins. To address these issues, described herein are novel recombinant fusion proteins that actively recruit linear DNA inserts in closer proximity to the genomic cleavage site, increasing integration efficiency of large DNA fragments into the genome. Such improvements to genomic editing technology allow one to use lower linear DNA concentrations without sacrificing efficiency and can be further combined with other features, such as fluorescent protein reporting systems.