Mutant AID Base Editor for Higher-Efficiency Single-Base Mutation
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Solution Overview
Problem
The existing CRISPR/Cas9-based single-base editing systems using activation-induced cytidine deaminase (AID) suffer from low mutation efficiency and large molecular weight, limiting their application in certain species and causing off-target effects.
Innovation Solution
A mutant protein of AID (AID10) is developed, combined with a DNA-specific binding protein, a uracil glycosylase inhibitor, and a nuclear localization signal, forming a high-efficiency base editor (HBE) that targets specific DNA regions with enhanced mutation efficiency and reduced molecular weight.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the existing CRISPR/Cas9-based single-base editing system using AID is used, then the system can achieve targeted base editing, but the molecular weight is large and mutation efficiency is low
Solution Approach 1:
The patent extracts and removes the Cas9 endonuclease component from the CRISPR/Cas9 system, retaining only the guide RNA (gRNA) for target recognition and the activation-induced cytidine deaminase (AID) for base editing. This extraction of the unnecessary Cas9 component significantly reduces the molecular weight of the editing system while maintaining the ability to achieve targeted single-base mutations through the gRNA-AID combination.
Solution Approach 2:
The patent optimizes the AID enzyme parameters by using truncated versions (AIDΔC18, AIDΔC35) that remove specific C-terminal regions, thereby reducing the molecular weight of the deaminase component. Additionally, the system uses modified gRNA structures and optimized fusion protein configurations to improve cellular delivery efficiency and mutation rates, directly addressing the productivity enhancement needed.
2Reliability
If the existing AID-based editing system is used, then the system can perform base deamination, but off-target effects occur
Solution Approach 1:
The patent introduces a uracil glycosylase inhibitor (UGI) as an intermediary component that blocks the uracil-DNA glycosylase (UDG) pathway. By inhibiting UDG, the system prevents the repair of uracil residues introduced by AID deamination, thereby forcing the cell to use error-prone translesion synthesis pathways that generate the desired mutations. This intermediary mechanism enhances specificity by controlling which repair pathways are active.
Solution Approach 2:
The patent employs a truncated AID enzyme (AIDΔC18 or AIDΔC35) with modified local properties at the C-terminus, which alters the enzyme's substrate specificity and reduces its activity at off-target sites. The localized modification of the AID structure improves the precision of deamination at the intended target site while minimizing unintended deamination events elsewhere in the genome.
3Ease of operation
If the existing AID-based editing system is used, then the system can convert cytosine to uracil, but the molecular weight limits transport to target DNA fragments
Solution Approach 1:
The patent segments the large Cas9-AID fusion protein into separate components: the guide RNA (gRNA) for target localization and a truncated AID deaminase (AIDΔC18 or AIDΔC35) for base editing. This segmentation allows each component to be optimized independently for cellular delivery, with the smaller AID fragment being more easily transported to the nucleus and delivered to target DNA fragments compared to the full-size Cas9-AID construct.
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 HBE system achieves higher mutation efficiency and specificity, enabling targeted single-base editing in various species, including mice, with improved stability and reduced off-target effects.
Implementation Method 1
AID10 is a mutant protein of activation-induced cytidine deaminase... cytidine deaminase converts cytosine base into uridine... DNA deaminase realizes the conversion of cytosine to thymine through deamination
Implementation Method 2
combined with a DNA-specific binding protein... targets specific DNA regions
Implementation Method 3
combined with... a uracil glycosylase inhibitor... when the uracil-DNA glycosylase pathway is inhibited, the system can also achieve more specific and desired point mutations
Implementation Method 4
combined with... a nuclear localization signal... enabling targeted single-base editing
Data Source
AI summary
The present application provides a mutant protein of activation-induced cytidine deaminase, wherein the mutant protein has the following mutations when compared to hsAID: T82I, K10E, K34E, E156G, 181*, S38C, H130R, V152A, R174G, and T110A. The present application also provides a high-efficiency base editor, including the mutant protein of activation-induced cytidine deaminase of the present application and a DNA-specific binding protein, which are linked sequentially via a linking sequence. The present application also provides a targeted single-base editing system, including a targeted single-base editing protein and a target hyper mutation fragment. Compared with the existing single-base editing system based on activation-induced cytidine deaminase (AID), the system for inducing the mutant protein of the present application has a smaller molecular weight and higher mutation efficiency.