Endonuclease–End-Processing Enzyme Coupling for Productive Gene Disruption
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Solution Overview
Problem
Current endonuclease platforms for targeted gene disruption suffer from low specificity, leading to off-target cleavage events and imprecise DNA repair, resulting in mutations and unproductive cleavage events.
Innovation Solution
Coupling site-specific endonucleases with end-processing enzymes to modify the DNA ends generated by cleavage, such as using exonucleases, polymerases, or phosphatases, to enhance the processing of DNA ends and increase the frequency of imprecise DNA repair events.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If endonucleases are used for targeted gene disruption, then site-specific DNA breaks can be generated, but off-target cleavage events occur due to low specificity
Solution Approach 1:
The patent combines endonucleases with end-processing enzymes into a single integrated system. The end-processing enzyme is coupled to the endonuclease such that it processes the DNA ends generated by the endonuclease cleavage, creating a synergistic effect that improves both specificity and disruption efficiency.
Solution Approach 2:
The end-processing enzyme acts as an intermediary that modifies the DNA ends between the endonuclease cleavage and the final gene disruption outcome. This intermediate processing step enhances the mutagenicity and ensures more precise gene targeting while reducing off-target effects.
2Productivity
If endonucleases generate double-strand DNA breaks, then gene disruption can be achieved, but imprecise DNA repair results in mutations
Solution Approach 1:
The end-processing enzyme performs preliminary processing of the DNA ends before the repair process occurs. By modifying the ends in advance (through exonucleolytic degradation, phosphorylation, or other processing), the system creates conditions that favor imprecise repair outcomes, thereby increasing gene disruption efficiency while controlling mutation types.
Solution Approach 2:
The coupling of end-processing enzymes changes the chemical and structural parameters of the DNA ends. This modification of end parameters (such as adding 5' phosphates, removing 3' overhangs, or creating specific chemistries) directs the repair machinery toward mutagenic outcomes, improving productivity while shaping the nature of mutations.
3Reliability
If conventional endonuclease platforms are used, then gene targeting is possible, but cleavage events are unproductive
Solution Approach 1:
The system ensures continuity of useful action by coupling the endonuclease cleavage directly with end-processing enzyme activity. The processing enzyme continuously acts on the generated ends to create conditions favorable for disruption, ensuring that every cleavage event has a high probability of being productive rather than reverting to precise repair.
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
Enhances the frequency of targeted gene disruption by improving the accuracy of DNA repair, reducing mutations and increasing the productivity of gene editing.
Implementation Method 1
site-specific endonucleases with end-processing enzymes to modify the DNA ends generated by cleavage, such as using exonucleases, polymerases, or phosphatases
Implementation Method 2
such as using exonucleases, polymerases, or phosphatases
Data Source
AI summary
The present disclosure relates to the co-expression of an endonuclease with an end-processing enzyme for the purpose of enhanced processing of the polynucleotide ends generated by endonuclease cleavage.