Cell-Free Base Editing Assay for Fast Mutation Screening
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Conducting genome editing experiments targeting mutated DNA in cells is challenging due to the difficulty in cell maintenance and the long time required, making it difficult to evaluate the efficiency of base editors effectively.
Innovation Solution
A cell-free test method involving contacting isolated double-stranded DNA with base-editing proteins in a single reaction tube, followed by sequencing to measure editing activity, allowing for the identification and quantification of target DNA mutations without complex cell-based experiments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If cell-based methods are used to evaluate base editing efficiency, then measurement accuracy is improved, but experimental time and complexity increase significantly
Solution Approach 1:
The patent creates a cell-free system that copies the essential functions of cellular base editing without requiring actual cells. By extracting and replicating the core components (base editor proteins, guide RNA, and DNA substrate) in a simplified in vitro environment, the method achieves accurate measurement of base editing efficiency while eliminating the time-consuming aspects of cell culture and maintenance
Solution Approach 2:
The patent extracts the essential base editing components from their natural cellular context and isolates them into a cell-free system. By separating the base editor protein, guide RNA, and DNA substrate from the complex cellular environment, the method maintains measurement accuracy while dramatically reducing experimental complexity and time requirements
2Reliability
If cell lines are used for genome editing experiments, then editing activity can be evaluated, but cell maintenance difficulty and experimental complexity increase
Solution Approach 1:
The patent creates a simplified copy of the cellular base editing system that retains all essential functions. By replicating only the necessary components (base editor, guide RNA, DNA substrate) in a cell-free environment, the method provides reliable base editor evaluation while eliminating the complex cell culture infrastructure and maintenance procedures
Solution Approach 2:
The patent extracts the core base editing machinery from the complex cellular system and isolates it into a manageable cell-free reaction system. This separation allows for reliable evaluation of base editing activity while dramatically simplifying the experimental procedure by removing cell culture, transfection, and cell handling steps
3Measurement precision
If cells from patients or animal models are used, then accurate mutation targeting is achieved, but cell isolation and maintenance become difficult
Solution Approach 1:
The patent creates a cell-free system that copies the essential function of targeting specific DNA mutations without requiring actual patient or animal cells. By using synthesized DNA substrates containing the target mutations and base editor proteins in a controlled in vitro environment, the method achieves accurate mutation targeting while eliminating the operational difficulties of cell isolation, culture, and maintenance
Solution Approach 2:
The patent extracts the DNA substrate containing the target mutation from the complex cellular material and isolates it as a purified component for use in the cell-free system. This extraction allows for accurate mutation targeting while completely eliminating the need for cell isolation and maintenance procedures
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
This method enables the measurement of base-editing activity and screening of base editors capable of correcting target DNA mutations in actual cells, with results comparable to cell-based methods, significantly reducing experimental time and complexity.
Implementation Method 1
base editing may employ a fusion protein in which a base-converting enzyme, such as a deaminase, is linked to nickase Cas9 (nCas9) or catalytically inactive Cas9 (dCas9), to induce single-base substitutions, such as A-to-G (adenine base editor) or C-to-T (cytosine base editor)
Data Source
Figure 1~3
Figure 4~6
Figure 7
AI summary
The present invention relates to a method for testing genome editing in a cell-free, in vitro system and, in particular, to a method for identifying and quantifying target DNA mutations by a base editor without using eukaryotic cells and without separate complicated experimental procedures. By using the present invention, target DNA mutations can be identified and quantified by a base editor without using cells and without separate complicated experimental procedures. In addition, a base editor having the ability to edit target DNA mutations in actual cells can be screened through a simple cell-free test method.