Split DddA Deaminase Base Editors for Mitochondrial Genome Editing
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Solution Overview
Problem
Current approaches to editing mitochondrial DNA (mtDNA) are limited by the challenge of transporting RNAs into mitochondria and the toxicity associated with delivering fully active DNA-binding proteins, which can cause cellular toxicity and are not suitable for homoplasmic mtDNA mutations or functionally recessive heteroplasmic mutations.
Innovation Solution
The development of a double-stranded DNA deaminase, DddA, and its variants, which can be split into inactive fragments for separate delivery and reconstitution within cells or mitochondria, combined with programmable DNA binding proteins to achieve precise base editing of mtDNA without inducing double-strand breaks, thereby minimizing toxicity and enabling editing of homoplasmic mutations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If fully active DNA-binding proteins are delivered to edit mtDNA, then editing efficiency is improved, but cellular toxicity increases
Solution Approach 1:
The DNA-binding protein is divided into multiple inactive fragments that cannot bind DNA individually. These fragments are delivered separately to the cell, then reconstitute into an active complex only at the target mtDNA site, thereby achieving editing efficiency while avoiding the toxicity of free active proteins in the cytoplasm
Solution Approach 2:
A mitochondrial targeting sequence acts as an intermediary that guides the reconstituted active complex into the mitochondria. This ensures that the active editing complex is formed only within the mitochondrial compartment, preventing off-target effects in the nucleus and cytoplasm while maintaining editing efficiency at the mtDNA target
2Adaptability or versatility
If RNA-based guide systems are used for CRISPR editing, then programming flexibility is improved, but delivery complexity increases
Solution Approach 1:
The guide RNA component is extracted from the CRISPR system and replaced with a simplified peptide-based targeting module. This removes the need for complex RNA delivery while retaining the ability to programmatically direct the editing complex to specific mtDNA sequences through customizable peptide sequences
Solution Approach 2:
The RNA-based programmable recognition system is replaced with a peptide-based system. Peptides are more stable and easier to deliver than RNA, and can be engineered to recognize specific DNA sequences through designed amino acid sequences, thereby simplifying delivery while maintaining programming flexibility
3Productivity
If double-strand breaks are induced to eliminate mutant mtDNA, then mutation elimination is improved, but mtDNA copy number decreases
Solution Approach 1:
Instead of using harmful double-strand breaks to eliminate mutant mtDNA, the invention uses precise base editing that converts the harmful mutation directly into the wild-type sequence. This approach eliminates the mutation while preserving the mtDNA molecule and maintaining copy number, thereby converting the harmful effect of mutation into a beneficial correction without the collateral damage of DSBs
4Reliability
If precision base editing is implemented, then therapeutic safety is improved, but technology complexity increases
Solution Approach 1:
The complex base editing system is segmented into modular components: inactive DNA-binding protein fragments, mitochondrial targeting sequences, and deaminase domains. Each module can be independently optimized and assembled, making the overall complex system more manageable and easier to engineer for specific therapeutic applications
Solution Approach 2:
The segmented DNA-binding protein fragments can be paired with different deaminase domains (e.g., APOBEC1, TadA) to create universal base editing platforms that can perform different types of edits (C-to-T, A-to-G). This multi-functionality reduces the need to develop entirely new systems for each editing type, thereby managing complexity while maintaining therapeutic safety
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 allows for precise and efficient installation of nucleotide changes in mtDNA, expanding the scope of genome editing technologies for modeling and therapeutic applications, including correcting pathogenic mutations without causing cellular toxicity.
Implementation Method 1
The DddA deaminase and variants thereof catalyze the deamination of double-stranded DNA
Data Source
AI summary
The specification provides programmable base editors that are capable of introducing a nucleotide change and/or which could alter or modify the nucleotide sequence at a target site in mitochondrial DNA (mtDNA) with high specificity and efficiency. Moreover, the disclosure provides fusion proteins and compositions comprising a programmable DNA binding protein (e.g., a mitoTALE, a mitoZFP, or a CRISPR/Casp) and double-stranded DNA deaminase that is capable of being delivered to the mitochondria and carrying out precise installation of nucleotide changes in the mtDNA. The fusion proteins and compositions are not limited for use with mtDNA, but also may be used for base editing of any double-stranded target DNA.