Adenine Base Editor Mutations Reduce Cytosine Editing
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Solution Overview
Problem
Adenine base editors (ABEs) suffer from off-target effects, including genome-level single-guide RNA-dependent off-target DNA editing and transcriptome-level sgRNA-independent off-target RNA editing, primarily due to incomplete target specificity and DNA/RNA-binding characteristics of adenosine deaminase, with insufficient focus on reducing cytosine editing activity.
Innovation Solution
Development of adenine base editors with specific mutations such as V106W, D108Q, F148A, and F149A in the adenosine deaminase enzyme to reduce cytosine editing activity while maintaining adenine editing efficiency, by fusing these mutations with CRISPR-associated protein 9 (Cas9) to enhance specificity and accuracy of adenine base editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If adenosine deaminase is used in adenine base editors, then adenine editing activity is achieved, but cytosine editing activity and RNA off-target effects occur
Solution Approach 1:
The patent applies parameter changes by introducing specific mutations (V106W, D108Q, F148A, F149A) in the adenosine deaminase enzyme to alter its catalytic properties. These mutations change the enzyme's substrate specificity parameters, reducing its ability to edit cytosine while maintaining adenine editing capability, thereby resolving the contradiction between achieving desired adenine editing and avoiding harmful cytosine editing and RNA off-target effects
Solution Approach 2:
The patent applies local quality by making specific localized mutations at particular amino acid positions (106, 108, 148, 149) within the adenosine deaminase structure. Each mutation locally modifies the enzyme's active site or substrate binding region, creating differential editing activities at specific locations while preserving overall enzyme function for adenine editing
2Object-generated harmful factors
If additional mutations are introduced to reduce RNA off-target effects, then RNA deamination activity decreases, but cytosine editing activity remains insufficiently addressed
Solution Approach 1:
The patent simultaneously addresses both RNA off-target effects and cytosine editing activity by introducing combinations of mutations that change multiple parameters of the adenosine deaminase enzyme. The D108Q mutation specifically reduces RNA deamination while the V106W, F148A, and F149A mutations collectively reduce cytosine editing activity, achieving comprehensive control over both harmful activities through coordinated parameter changes
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
The modified adenine base editors exhibit improved specificity for adenine editing with significantly reduced cytosine editing activity and RNA off-target effects, demonstrating enhanced accuracy and efficiency in adenine base editing.
Implementation Method 1
Adenine base editors (ABEs) are effective gene editing tools that can convert an A/T pair to a G/C pair
Data Source
AI summary
The present invention relates to an adenine base editor from which cytosine editing activity is removed, a method of editing an adenine base, and method of editing an adenine base. In the present invention, it was confirmed that four mutations (V106W, D108Q, F148A and F149A) in adenosine deaminase increase the specificity for adenine editing by reducing cytosine base editing efficiency, and ABE variants were manufactured by introducing respective mutations into a variety of more improved adenine base editors than ABEmax, and tested, confirming that the mutations significantly decrease a cytosine editing effect. Therefore, the adenine base editor according to the present invention is able to more accurately edit adenine, and the method of editing an adenine base can be effectively used in the field of gene therapy that has to accurately edit only adenine in all living organisms including humans, plants and bacteria or novel crop development.