Adenosine Deaminase Mutations for Precise Adenine Base Editing
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Solution Overview
Problem
Current base editors have varying site-dependent editing efficiencies and a wide editing window, leading to unnecessary editing, thus necessitating improvements for more precise and efficient genome editing.
Innovation Solution
A site-directed mutation is performed on the basic sequence of the adenosine deaminase to create an adenine base editor with improved editing efficiency, and a suitable position for embedding adenosine deaminase in the nuclease domain of the base editor fusion protein is identified to enhance editing efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If current base editors are used, then genome editing can be achieved, but editing efficiency varies by site and editing window is wide causing unnecessary editing
Solution Approach 1:
The patent applies local quality by introducing a narrow editing window through specific amino acid mutations in the adenosine deaminase domain (positions 104-107 and 148-155). These localized mutations create a more constrained editing window that maintains high editing efficiency at target sites while preventing off-target editing, thus simultaneously improving productivity and manufacturing precision.
Solution Approach 2:
The patent changes key parameters of the adenosine deaminase enzyme through site-directed mutations at specific positions (104V107 and 148-155). These parameter changes in the enzyme's amino acid sequence result in a narrowed editing window and enhanced site-specific editing efficiency, resolving the contradiction between overall editing productivity and precision.
2Productivity
If adenosine deaminase is embedded in nuclease domain at certain positions, then base editing efficiency is improved, but protein structure stability may be affected
Solution Approach 1:
The patent segments the base editor fusion protein into distinct functional domains: the nuclease domain (Cas9) and the adenosine deaminase domain. By maintaining this segmentation and placing the deaminase at specific positions (104V107 and 148-155) rather than disrupting the overall protein architecture, the patent achieves improved base editing efficiency while preserving protein structure stability through modular domain organization.
Solution Approach 2:
The patent uses a linker sequence as an intermediary between the nuclease domain and adenosine deaminase domain. This linker acts as a flexible connector that allows the deaminase to be positioned at optimal locations for enhanced editing efficiency while maintaining proper protein folding and structural stability through the mediating linker region.
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 base editor achieves significantly higher editing efficiency, particularly on the PCSK9 target and other sites, with great potential for treating diseases associated with point mutations, such as hypercholesterolemia, transthyretin amyloidosis, and β-hemoglobinopathy.
Implementation Method 1
deaminates, under guidance of sgRNA, a target base adenine A located in a base editing active window to form hypoxanthine I
Data Source
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AI summary
The present invention belongs to the field of biotechnology, and provides a deaminase and an adenine base editor, and also provides a mutated deaminase and a corresponding adenine base editor. The mutated deaminase undergoes mutation of a plurality of amino acids compared with a parent deaminase, thereby improving a base editing efficiency and obtaining a good application prospect.