Split Deaminase Base Editing for Plant Organelle DNA
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current genetic editing tools, such as CRISPR systems, are not suitable for editing DNA sequences in plant organelles like mitochondria and chloroplasts due to difficulties in delivering guide RNAs and co-expressing necessary compounds within these organelles.
Innovation Solution
A composition and method for editing adenine to guanine in plant cell organelle DNA, utilizing a DNA binding protein, split cytosine deaminase, and adenine deaminase, which allows for targeted base editing without generating DNA double-strand breaks.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR systems or conventional base editing tools are used, then genome editing capability is achieved, but delivery of guide RNAs and co-expression of necessary compounds in plant organelles becomes difficult
Solution Approach 1:
The deaminase enzyme is divided into two inactive halves (first deaminase half and second deaminase half) that can be independently delivered to organelles. Each half alone cannot perform deamination, but when both are present in the organelle, they reassemble into functional deaminase activity, enabling base editing without requiring guide RNA delivery or complex co-expression systems
Solution Approach 2:
The DNA binding protein acts as an intermediary that delivers and positions the split deaminase halves at the target site within the organelle. The DNA binding protein binds to specific DNA sequences and recruits the deaminase halves, enabling targeted editing without requiring guide RNA molecules
2Reliability
If full-length cytosine deaminase is used, then deamination activity is achieved, but toxicity to host cells occurs
Solution Approach 1:
The cytosine deaminase is segmented into two inactive halves that cannot perform deamination individually. When delivered separately to host cells, these inactive halves avoid toxicity because they lack catalytic activity. Only when both halves are present do they reassemble into functional deaminase, providing controlled activity reduction of toxicity
Solution Approach 2:
Instead of delivering full-length active deaminase that causes toxicity, only partial (inactive) versions are delivered. The system provides just enough activity when the two halves combine, avoiding excessive deamination activity that would harm host cells
3Productivity
If adenine deaminase is delivered as full-length protein, then base editing efficiency is improved, but off-target effects increase
Solution Approach 1:
The adenine deaminase is divided into two inactive halves that must reassemble at the target site to become active. This spatial control ensures that deamination activity occurs only where both halves are present together - at the intended target location - thereby reducing off-target effects while maintaining on-target editing efficiency
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 adenine base editing in plant organelle DNA, facilitating the development of herbicide-resistant, spectinomycin-resistant, and albino phenotype plants, while reducing off-target effects.
Implementation Method 1
an adenine deaminase or a nucleic acid encoding the same
Implementation Method 2
a DNA binding protein or a nucleic acid encoding the same
Data Source
Figure 1
Figure 2a~2c
Figure 3a~3b
AI summary
The present invention relates to a composition for base editing of plant cell organelle DNA and, in particular, to a composition and editing method for adenine-to-guanine editing of plant cell organelle DNA.