Real-time Base Editing Reporter Systems for Fluorescence Monitoring
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Solution Overview
Problem
Current methods for monitoring base editing in living cells, such as CRISPR technology, rely on DNA sequencing which is cumbersome, expensive, and not suitable for screening and optimization of single base editing frequencies.
Innovation Solution
Development of a real-time reporter system, referred to as AMBER or ACE, utilizing APOBEC-Cas9 fusions that enables fluorescence-based monitoring of base editing by restoring fluorescence in reporter proteins like mCherry and eGFP, allowing for quantification of editing frequencies and DNA cleavage activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If DNA sequencing is used to monitor base editing, then measurement precision is achieved, but device complexity and loss of time increase
Solution Approach 1:
The patent replaces the mechanical/biochemical process of DNA sequencing with a fluorescence-based optical detection system. The base editing event is coupled to fluorescence signal changes through the use of fluorescent proteins (eGFP, mCherry) whose expression or conformational state changes in response to the editing event, allowing real-time monitoring without sequencing
Solution Approach 2:
The patent creates a fluorescent copy or surrogate readout of the base editing event. Instead of directly detecting the nucleotide change, the system uses fluorescent protein expression levels or fluorescence resonance energy transfer (FRET) signals that replicate the editing information in a easily measurable format
2Measurement precision
If DNA sequencing is used to monitor base editing, then measurement precision is achieved, but device complexity and cost increase
Solution Approach 1:
The patent replaces complex sequencing infrastructure with simple fluorescence microscopy or plate reader equipment. The detection system uses standard fluorescent protein markers that can be visualized with conventional optical equipment, eliminating the need for expensive sequencing machines and complex data analysis pipelines
Solution Approach 2:
The patent utilizes color changes in fluorescent proteins as a direct readout of base editing events. Different fluorescent proteins (eGFP for green, mCherry for red) or fluorescence intensity changes provide visual and quantitative information about editing efficiency, allowing rapid assessment without complex analytical equipment
3Measurement precision
If DNA sequencing is used to monitor base editing, then measurement precision is achieved, but ease of operation deteriorates
Solution Approach 1:
The patent replaces the labor-intensive DNA extraction, sequencing library preparation, and bioinformatics analysis workflow with a simple fluorescence measurement protocol. Researchers can directly visualize and quantify editing events in living cells or cell lysates using standard fluorescence detection methods, dramatically simplifying the operational process
Solution Approach 2:
The patent implements self-reporting base editing through fluorescent protein markers that automatically indicate editing status. The system uses self-cleaving peptides (T2A) to couple fluorescent protein expression to the editing event, creating a self-contained reporting mechanism that requires no external intervention or complex processing
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 provides a rapid, efficient, and quantitative assay for base editing, enabling real-time monitoring and optimization of base editing technologies with minimal off-target effects, and is applicable across various cell types.
Implementation Method 1
an apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like-1 (APOBEC1) single-stranded DNA deaminase
Implementation Method 2
restoring fluorescence to a first reporter (e.g., enhanced green fluorescent protein; eGFP) marked by a second reporter (e.g., mCherry)
Data Source
AI summary
Real-time systems for monitoring base editing in living cells, including base editing by APOBEC-Cas9 fusions, is provided herein.