Adenine Base Editing with DNA Glycosylase for A-to-C and A-to-T Transversions
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Solution Overview
Problem
Current genome editing technologies, such as cytosine and adenine base editors, are unable to efficiently correct human pathogenic point mutations that require A-to-C and A-to-T transversions, which account for nearly a quarter of human disease-associated point mutations, particularly the transversion of A⋅T to C⋅G, which is the second most common pathogenic SNV.
Innovation Solution
A base editing system is constructed by fusing 3-methyladenine DNA glycosylase with adenosine deaminase and Cas9 nuclease with impaired catalytic activity to achieve adenine-based transversion, specifically targeting adenine to cytosine or thymine mutations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If classic base editors (CBEs or ABEs) are used, then base conversion efficiency is improved, but the ability to achieve A-to-C and A-to-T transversions is lost
Solution Approach 1:
The patent combines three functional components into a single base editor system: Cas9 nickase (for DNA binding and nicking), adenosine deaminase TadA (for A-to-I deamination), and 3-methyladenine DNA glycosylase (for excising the damaged base and enabling transversion). This merging allows the system to achieve both base conversion and A-to-C/A-to-T transversions that neither CBE nor ABE could accomplish alone
Solution Approach 2:
The base editor is constructed as a composite protein system with multiple enzymatic activities fused together. The Cas9 nickase provides targeting specificity, TadA provides deamination function, and the 3-methyladenine DNA glycosylase provides the transversion capability. This composite structure enables the system to perform functions that individual components cannot achieve
2Reliability
If traditional genome editing technology mediated by homologous recombination is used, then base mutation correction is achieved, but editing efficiency is very low (0.1% to 5%)
Solution Approach 1:
The patent replaces the complex homologous recombination mechanism with a simpler enzymatic pathway. Instead of relying on cellular recombination machinery to insert donor templates, the system uses engineered enzymes (Cas9 nickase + TadA + 3-methyladenine DNA glycosylase) to directly convert the target base through deamination and excision, followed by error-prone repair that favors transversion outcomes. This substitution dramatically increases editing efficiency from 0.1-5% to significantly higher rates
3Productivity
If ABE is used for base editing, then editing efficiency is improved (53% for A⋅T-G⋅C), but the ability to correct A-to-C and A-to-T pathogenic mutations is lost
Solution Approach 1:
The patent modifies the local enzymatic properties of the base editing system by incorporating 3-methyladenine DNA glycosylase, which has specific affinity for hypoxanthine (the deaminated product of adenine). This localized enzymatic enhancement at the target site enables the system to perform A-to-C and A-to-T transversions while maintaining the high efficiency provided by TadA's deamination activity
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 system achieves high editing efficiency for A-to-C and A-to-T mutations, correcting up to 23.4% of A⋅T to C⋅G and 12% of A⋅T to T⋅A mutations, addressing a significant portion of human disease-associated point mutations.
Implementation Method 1
adenine base editor ABE7.10 that can act on single stranded DNA is finally obtained... an active editing region is mainly located at positions 4-7. The average editing efficiency of A⋅T-G⋅C caused by this system
Implementation Method 2
3-methyladenine DNA glycosylase with impaired activity... the substitution of C⋅G-T⋅A is achieved within a range of 20 bp of an upstream targeting sequence of NGG
Implementation Method 3
a Cas9 protein recognizes and specifically binds to DNA using NGG as PAM
Implementation Method 4
under the action of deaminase and DNA repair, finally, the substitution of C⋅G-T⋅A is achieved
Data Source
AI summary
A base editing system for achieving A-to-C and/or A-to-T base mutations and a use thereof are provided. A base editor is constructed by means of fusing 3-methyladenine DNA glycosylase with adenosine deaminase and Cas9 nuclease with impaired catalytic activity, which achieves adenine-based transversion for the first time. It is found through experimental comparison that AXBE, which is constructed by means of fusing mouse-derived 3-methyladenine DNA glycosylase with adenosine deaminase TadA-8e derived from E. coli and Cas9 nickase with impaired catalytic activity derived from Streptococcus pyogenes, has the best effect of catalyzing the transversion of adenine. The use of the base editing system in the gene therapy, cell therapy, human disease model production, and crop genetic breeding, etc. is promoted.