Mitochondrial DNA Editing With Block-and-Nick Deletions
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Solution Overview
Problem
Current methods for editing mitochondrial DNA (mtDNA) are inefficient and imprecise, leading to challenges in creating stable animal models of severe mitochondrial diseases and understanding the role of mtDNA in health and disease, as traditional genome engineering tools like TALE-nucleases and CRISPR-Cas9 primarily rely on double-strand break repair, which can trigger rapid degradation and are inefficient in mitochondria.
Innovation Solution
A 'block and nick' DNA modification system using sequence-specific TALE proteins flanking a single-strand DNA nick on the light strand of mtDNA to induce targeted deletions with enhanced precision, allowing for predictable deletions at specific regions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional genome engineering tools (CRISPR-Cas9, TALE nucleases) are used to edit mtDNA, then double-strand breaks can be introduced to combat heteroplasmy, but both NHEJ and HDR are very inefficient and cannot achieve precise sequence engineering
Solution Approach 1:
The invention segments the DNA cleavage function into two separate components: a nickase that creates single-strand nicks and blocking molecules that prevent repair. This segmentation allows controlled induction of deletions without requiring efficient double-strand break repair, resolving the contradiction between editing efficiency and precision in mtDNA
Solution Approach 2:
Instead of using double-strand breaks to combat heteroplasmy (traditional approach), the invention inverts the strategy by using single-strand nicks combined with blocking molecules to induce targeted deletions. This inversion achieves precise sequence engineering while maintaining reliability, as the blocking molecules ensure the nicked strand cannot be repaired
2Productivity
If DSBs are used strategically to combat heteroplasmy in patient cells, then some repair can occur at low frequencies, but NHEJ and HDR are very inefficient and have not been useful for mtDNA sequence engineering
Solution Approach 1:
The invention applies preliminary action by first introducing a single-strand nick at a specific location in mtDNA, then introducing blocking molecules that prevent repair at that site. This preliminary nicking followed by blocking ensures that when deletions occur, they are precisely targeted and engineered, making the low-frequency repair events useful for sequence engineering rather than random
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 method achieves highly predictable and stable deletions in mtDNA, enabling the creation of animal models of mitochondrial diseases with targeted deletions maintained through adulthood and applicable to human cells, facilitating research on mtDNA function and disease modeling.
Implementation Method 1
two mitoTALE proteins serving as blocks flanking a single-stranded DNA nick on the light strand of mtDNA
Implementation Method 2
a DNA cleaving enzyme that generates a single strand nick on the light strand of the mtDNA sequence to be deleted
Data Source
AI summary
Disclosed is a method for editing mitochondrial DNA (mtDNA) within a cell, which include introducing into the cell (a) a DNA cleaving enzyme targeted to the mtDNA sequence to be deleted; (b) a first DNA binding component targeted to a sequence adjacent to the 5′ end of a mtDNA sequence to be deleted; and (c) a second DNA binding component targeted to a sequence adjacent to the 3′ end of the mtDNA sequence to be deleted, where the DNA cleaving enzyme generates a double stranded break (DSB) within the mtDNA sequence to be deleted or generates a single strand nick on the light strand of the mtDNA sequence to be deleted, and wherein the mtDNA sequence between the target sequence for the first DNA binding component and the target sequence for the second DNA binding component is deleted.