Cytosine Base Editing System Loop Mutations
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Solution Overview
Problem
The cytosine base editing system suffers from unpredicted off-target effects and lacks specificity due to overexpression of cytosine deaminase, often resulting in multiple C mutations instead of a single targeted mutation, affecting its accuracy and application in gene editing.
Innovation Solution
Optimization of Loop1 and Loop7 in the human-derived hA3Bctd domain of APOBEC3B C-terminal domain to alter the binding ability of cytosine deaminase to single-stranded DNA, reducing off-target effects and enhancing specificity and accuracy through mutation testing in rice protoplast transformation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If cytosine deaminase is overexpressed in the genome to enhance base editing efficiency, then the deamination ability is improved, but random off-target deamination occurs in high transcriptional active regions
Solution Approach 1:
The patent applies parameter changes by modifying the binding affinity parameters of cytosine deaminase to single-stranded DNA. Through mutagenesis of specific residues (e.g., R211K, R311K, Y313F, D314R), the binding strength is tuned to reduce off-target effects while preserving on-target editing efficiency. This resolves the contradiction by changing the biochemical parameters of the enzyme rather than its expression level.
Solution Approach 2:
The patent applies local quality by creating heterogeneous binding properties at different genomic locations. The modified cytosine deaminase exhibits differential binding affinity: weak binding to transient single-stranded DNA in high-transcription regions (reducing off-target effects) while maintaining sufficient binding at the targeted site guided by gRNA-Cas9 complex. This local differentiation resolves the contradiction between efficiency and specificity.
2Productivity
If the binding ability of cytosine deaminase to single-stranded DNA is enhanced to improve deamination efficiency, then the editing productivity increases, but the editing window becomes wider causing multiple C mutations
Solution Approach 1:
The patent applies parameter changes by precisely tuning the binding affinity parameter of cytosine deaminase to single-stranded DNA through point mutations. The modified enzyme has reduced binding strength compared to wild-type, which narrows the editing window to primarily affect only the targeted cytosine while minimizing deamination of adjacent cytosines. This resolves the contradiction between efficiency and precision.
Solution Approach 2:
The patent applies partial action by using a cytosine deaminase with moderately reduced binding affinity rather than complete loss of binding. This partial reduction is sufficient to narrow the editing window and prevent multiple C mutations, while still allowing effective deamination at the intended target site. The principle of partial action resolves the contradiction by finding the optimal intermediate state.
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 editing system achieves high-efficiency, high-specificity, and high-accuracy base editing with a narrowed editing window, significantly reducing genome-wide off-target effects and improving the precision of cytosine base editing.
Implementation Method 1
a single-stranded DNA region is formed by the Cas9 single-stranded nickase
Implementation Method 2
the deaminase can efficiently and respectively remove amino groups of C and A nucleotides on single-stranded DNA at a targeting position to become U base
Data Source
AI summary
The present invention belongs to the field of gene editing. In particular, the present invention relates to an improved cytosine base editing system which has a significantly reduced genome-wide off target effect and a narrow editing window.