I-TevI Chimeric Endonuclease Monomeric Cleavage Specificity
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Solution Overview
Problem
Current gene editing technologies, such as those using FokI nuclease domains, require dimerization for efficient DNA cleavage, necessitating dual target sites and spacers, which can lead to accidental off-site cleavage and reduced specificity.
Innovation Solution
Development of monomeric chimeric endonucleases using the GIY-YIG homing endonuclease I-TevI, which does not require dimerization, and identification of new preferential cleavage sites to enhance specificity and efficiency of nucleic acid targeting.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If FokI nuclease domain is used for gene editing, then DNA cleavage efficiency is improved, but specificity deteriorates due to requirement of dimerization and dual target sites
Solution Approach 1:
The patent extracts the dimerization requirement from the FokI nuclease system by using a single homing endonuclease molecule that performs both DNA binding and cleavage functions independently, eliminating the need for dual target sites and spacers that cause off-site cleavage
Solution Approach 2:
The homing endonuclease I-TevI serves multiple functions within a single protein: it binds to the target DNA sequence through its DNA-binding domain and simultaneously performs cleavage through its catalytic domain, replacing the need for separate FokI dimerization system
2Productivity
If dual target sites and spacers are used with FokI, then cleavage efficiency is improved, but device complexity increases
Solution Approach 1:
The patent removes the complex spacer and dual target site architecture from the system by employing a single homing endonuclease that recognizes and cleaves its target independently, simplifying the overall gene editing design
3Reliability
If monomeric chimeric endonucleases are used, then specificity is improved by reducing off-site cleavage, but cleavage efficiency may deteriorate
Solution Approach 1:
The patent optimizes the parameters of the homing endonuclease I-TevI, including its catalytic domain configuration and recognition sequence characteristics, to achieve both high specificity and maintained cleavage efficiency in monomeric form
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
The use of I-TevI chimeric endonucleases with optimized cleavage sites improves the specificity and efficiency of gene editing by reducing accidental cleavage events and increasing the number of targetable nucleic acid sequences.
Implementation Method 1
The present invention relates to a method to cleave target nucleic acid sequence by the catalytic domain of a GIY-YIG homing endonucleases I-TevI
Data Source
AI summary
The present invention relates to a method to cleave target nucleic acid sequence by the catalytic domain of a GIY-YIG homing endonucleases I-TevI. More precisely, the invention relates to the deciphering of new preferential I-TevI cleavage sites for efficient and specific cleavage activity. The invention concerns a method for the generation of TevI specific chimeric endonucleases to target nucleic acid sequence including such cleavage sites and methods of using same for gene editing.