Plant Organelle Base Editing With Split Deaminase Composition
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Solution Overview
Problem
Existing genome editing tools are not suitable for editing DNA sequences in plant organelles such as mitochondria and chloroplasts due to difficulties in delivering guide RNAs and expressing components simultaneously, which is necessary for studying gene function and improving crop traits.
Innovation Solution
A composition comprising a DNA binding protein, split cytosine deaminase, and adenine deaminase is used to perform A-to-G and C-to-T base editing in plant organelle DNA, enabling simultaneous editing of adenine to guanine and cytosine to thymine.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional genome editing tools (CRISPR, ZEN, TALEN) are used, then genome editing capability is achieved, but they cannot edit plant organelle DNA due to difficulties in delivering guide RNAs and expressing components simultaneously
Solution Approach 1:
The cytosine deaminase is divided into two inactive split forms (first split and second split) that can be independently delivered and expressed. When both splits are present in the organelle, they complement each other to form the active deaminase enzyme, enabling C-to-T editing. This segmentation allows separate delivery of components that would otherwise be difficult to co-deliver as a single complex.
Solution Approach 2:
The invention uses split deaminase components as intermediaries that require each other for functionality. The first split deaminase and second split deaminase act as complementary intermediaries that only form an active editing complex when both are present in the target organelle, providing a built-in mechanism to ensure proper assembly and reduce off-target effects.
2Manufacturing precision
If DddAtox cytosine deaminase is used for C-to-T editing, then base editing capability is achieved, but it causes toxicity in host cells
Solution Approach 1:
The toxic DddAtox cytosine deaminase is segmented into two inactive portions (first split and second split). Each split alone is non-toxic and non-functional, but when both are present in the host cell, they complement to form the active deaminase enzyme. This segmentation strategy maintains editing precision while eliminating toxicity by ensuring the active enzyme is only formed under controlled conditions.
Solution Approach 2:
The invention converts the harmful toxic property of DddAtox into a beneficial controlled activation mechanism. The toxicity is eliminated by splitting the enzyme, and the same splitting mechanism enables controlled reactivation only when both components are present in the target organelle, transforming a harmful characteristic into a safety feature.
3Productivity
If single deaminase components are used, then individual base editing (A-to-G or C-to-T) is achieved, but simultaneous dual base editing cannot be performed
Solution Approach 1:
The invention merges multiple deaminase functionalities (adenine deaminase for A-to-G editing and split cytosine deaminase for C-to-T editing) into a single composition. The composition includes DNA binding protein, first split deaminase, second split deaminase, and adenine deaminase, all working together in the same organelle to perform dual base editing simultaneously, thereby increasing editing throughput without requiring separate transformation events.
Solution Approach 2:
The composition is designed with multi-functionality to perform both A-to-G and C-to-T base editing within the same organelle. The DNA binding protein can target specific sequences, while the combined deaminase components provide dual enzymatic activities, making the composition universally applicable for various editing needs in plant organelles.
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 the development of functional plants with traits like herbicide resistance by effectively editing plant organelle DNA, which was previously impossible.
Implementation Method 1
Bacterial toxin DddAtox is an enzymatic moiety in bacterial toxins derived from Burkholderia cenocepacia and is responsible for cytosine deamination within double-stranded DNA
Data Source
AI summary
The present invention relates to a composition for base editing of plant cell organelle DNA and, in particular, to a composition and method for editing adenine to guanine and cytosine to thymine in plant cell organelle DNA.