Mitochondrial Genome Editing via AAV-delivered CRISPR
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Solution Overview
Problem
Current methods are inadequate for effectively screening potential therapeutics for mitochondrial disorders caused by pathogenic mtDNA mutations, particularly due to the lack of reliable mitochondrial disease models and understanding of the pathophysiology, as seen in conditions like MELAS syndrome.
Innovation Solution
A method involving the use of a CRISPR system, specifically a Cas9 protein and guide RNA, delivered via mitochondria-specific vectors or adeno-associated viruses, to edit or regulate mitochondrial DNA, enabling site-specific cutting or the introduction of transcriptional regulators to modulate gene expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR system is delivered to mitochondria using mitochondrial targeting sequences, then editing precision is improved, but delivery complexity increases
Solution Approach 1:
The CRISPR system is divided into separate components: Cas9 protein, guide RNA, and mitochondrial targeting sequence. These components can be delivered separately or as pre-assembled complexes, allowing optimization of each component's delivery while maintaining overall precision.
Solution Approach 2:
Mitochondrial targeting sequences act as intermediaries that guide the CRISPR components to the correct destination. These sequences serve as molecular addresses that direct Cas9 and guide RNA to mitochondrial DNA without requiring complex delivery mechanisms.
2Reliability
If Cas9 protein and guide RNA are used to cut mitochondrial DNA, then disease model creation is improved, but off-target effects increase
Solution Approach 1:
The system uses transient expression of Cas9 and guide RNA to achieve sufficient editing for disease model creation without prolonged exposure that would increase off-target effects. The temporary presence of the editing machinery limits collateral damage while maintaining effectiveness.
Solution Approach 2:
The patent uses guide RNA sequences that are copied from designed templates with high fidelity. This ensures precise matching to the intended target site and reduces the likelihood of off-target binding and unintended edits.
3Adaptability or versatility
If mitochondrial DNA is edited to create disease models, then drug discovery capability is improved, but time required for model generation increases
Solution Approach 1:
The CRISPR components are prepared and validated in advance before being applied to create specific disease models. Guide RNAs are designed and tested for specificity, and Cas9 variants are pre-selected for optimal activity, reducing the time needed during actual model generation.
Solution Approach 2:
The system allows rapid adjustment of editing parameters such as guide RNA sequence, Cas9 variant, and transfection conditions to optimize for different disease models. This flexibility enables quick adaptation to various mitochondrial disorders without starting from scratch each time.
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 allows for precise editing or regulation of mitochondrial DNA, potentially creating disease models and facilitating drug discovery by altering target mitochondrial sequences, thereby addressing the challenges in treating disorders like MELAS syndrome.
Implementation Method 1
Bacterial and archaeal CRISPR-Cas systems rely on short guide RNAs in complex with Cas proteins to direct degradation of complementary sequences present within invading foreign nucleic acid
Data Source
AI summary
Methods and compositions of altering mitochondrial DNA of a eukaryotic cell are provided using one or more of a mitochondrial specific adeno-associated virus to deliver one or more nucleic acids encoding CRISPR system including a Cas9 protein or its nuclease inactive variant and a guide RNA into a mitochondria for expression within the mitochondria. The Cas9 system can cut, nick or regulate a target mitochondrial nucleic acid.