Frameshift Mutation Correction via Blunting Enzyme and Nuclease
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Solution Overview
Problem
Current genetic treatments for frameshift mutations, caused by insertion or deletion of nucleotides in DNA, are inefficient and often result in undesired side effects due to the need for removing large sections of genome sequences.
Innovation Solution
A composition comprising a target-specific nuclease, such as Cas9, combined with a double-strand break-end blunting enzyme, guided by a guide RNA, to induce precise and predictable mutations by creating staggered or blunt ends in DNA, allowing for accurate insertion or deletion of single base pairs without the use of a donor template.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If large sections of genome sequence are removed to treat frameshift mutations, then the mutation can be corrected, but undesired side effects occur and treatment efficiency is reduced
Solution Approach 1:
The treatment approach is segmented into two distinct components: (1) a target-specific nuclease (e.g., Cas9) that creates a precise double-strand break at the mutation site, and (2) a double-strand break-end blunting enzyme that processes the break to enable accurate repair. This segmentation allows each component to perform its specialized function, achieving both precision and efficiency that neither component could achieve alone.
Solution Approach 2:
The double-strand break-end blunting enzyme acts as an intermediary between the nuclease-induced break and the cell's natural repair mechanisms. By processing the break ends to create blunt or staggered ends, this intermediary enables the repair machinery to accurately reconstruct the sequence without requiring large genomic deletions, thus improving both safety and efficiency.
2Manufacturing precision
If precise and predictable mutations are induced by creating staggered or blunt ends, then frameshift mutations can be corrected accurately, but the device complexity increases due to multiple enzyme components
Solution Approach 1:
Multiple functional components are merged into a single composition: the target-specific nuclease, the double-strand break-end blunting enzyme, and the guide RNA work together as an integrated system. This merging allows the complex function of precise genome editing to be achieved through coordinated action of specialized components, each optimized for its specific task.
Solution Approach 2:
The composition is designed with universal applicability through the use of guide RNA, which can be programmed to direct the nuclease to any target sequence with appropriate PAM sites. The same composition architecture can correct different frameshift mutations at various genomic locations by simply changing the guide RNA sequence, reducing the need for multiple specialized treatments.
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 enables efficient and precise correction of frameshift mutations, reducing the risk of side effects and achieving predictable genome editing in human cells, thereby treating diseases caused by such mutations.
Implementation Method 1
a target specific nuclease... cleaving the dsDNA at a target site with a target specific nuclease, wherein the cleavage results in overhangs on both dsDNA ends
Implementation Method 2
a double strand break (DSB)-end blunting enzyme... inserting a nucleotide complementary to the overhanging nucleotide on both of the dsDNA ends using a double strand break (DSB)-end blunting enzyme, or removing the overhanging nucleotide on both of the dsDNA ends using the DSB-end blunting enzyme
Implementation Method 3
The target specificity of the nuclease can be provided by a guide RNA (gRNA)... The gRNA can be a single guide RNA (sgRNA)
Implementation Method 4
and ligating the dsDNA ends together, thereby inserting or deleting a single base pair in the dsDNA
Data Source
AI summary
The disclosure provides systems, methods, and compositions for a target specific nuclease and a blunting enzyme to correct frameshift mutations for genome editing and treatment of diseases. In some embodiments, the target specific nuclease and the blunting enzyme are combined with a guide RNA and/or a microhomology-mediated end joining (MMEJ) inhibitor.