Gene Editing Module Analysis Using DPH Toxin Selection and HRM PCR
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Solution Overview
Problem
Existing gene editing technologies, particularly CRISPR/Cas9 systems, face challenges in differentiating between homozygous and heterozygous gene inactivation and site-specific versus non-specific integration events, often leading to off-target mutations and genomic instability, and lack robust methods for large-scale, statistical analysis.
Innovation Solution
A method combining gene inactivation with toxin selection and antibiotic resistance, utilizing DPH gene transcription-sensitive toxins like pseudomonas exotoxin and diphtheria toxin, coupled with high-resolution melting (HRM) PCR, allows for the differentiation and quantification of homozygous and heterozygous modifications, as well as site-specific and non-specific integration events, enabling efficient and specific gene editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If CRISPR/Cas9 systems are used for gene editing, then gene editing efficiency is improved, but off-target mutations and genomic instability increase
Solution Approach 1:
The patent employs a dual-selection marker system that provides feedback mechanisms to monitor and control gene editing outcomes. The first selection marker identifies cells that have undergone any integration event, while the second marker specifically identifies homozygous inactivation events, enabling researchers to select for desired editing outcomes and eliminate off-target effects through iterative selection rounds.
Solution Approach 2:
The patent changes the selection parameters by using two different selection markers with distinct selection pressures. This allows for staged selection processes where cells are first selected for integration events, then further selected for homozygous inactivation, thereby optimizing the editing outcome while minimizing off-target effects through controlled parameter changes in the selection process.
2Productivity
If existing gene editing methods are used, then gene inactivation is achieved, but differentiation between homozygous and heterozygous modifications is not possible
Solution Approach 1:
The patent introduces a second selection marker as an intermediary tool that specifically reports on the zygosity status of gene inactivation events. This intermediary marker system enables the differentiation between homozygous and heterozygous modifications without interfering with the primary gene inactivation process, allowing precise measurement and selection of desired genetic outcomes.
3Productivity
If gene editing is performed without site-specific verification, then integration events occur, but site-specific versus non-specific integration cannot be distinguished
Solution Approach 1:
The patent implements a feedback-based verification system where the second selection marker provides information about the nature of integration events. By monitoring which cells express the second marker, researchers can distinguish between site-specific homozygous inactivation events and non-specific integration events, enabling selective amplification of desired editing outcomes.
4Quantity of substance
If large-scale statistical analysis of gene editing is performed, then comprehensive data is obtained, but existing methods lack robustness for such analysis
Solution Approach 1:
The patent creates a universal selection system that can be applied across large numbers of cells and different gene editing scenarios. The dual-marker system provides multi-functional capability by simultaneously identifying integration events, homozygous inactivation, and enabling statistical analysis, making the method robust and scalable for comprehensive gene editing evaluation.
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
Enables high-throughput analysis of gene editing efficiency and specificity by distinguishing between different integration and inactivation events, facilitating the optimization of CRISPR/Cas9 systems and other gene editing modules, reducing off-target effects and improving therapeutic applications.
Implementation Method 1
transfected cells are cultivated in the presence of a DPH gene transcription sensitive toxin
Implementation Method 2
coupled with high-resolution melting (HRM) PCR
Data Source
AI summary
Herein is reported a method for determining the introduction of a nucleic acid into the genome of a mammalian cell, whereby the mammalian cell comprises one or two transcriptionally active alleles of a DPH1, DPH2, DPH4 and/or DPH5 gene, comprising the steps of transfecting the mammalian cell with one or more plasmids comprising the nucleic acid to be introduced, and the elements required for gene editing of said DPH gene, cultivating the transfected cell in the presence of a DPH gene transcription sensitive toxin, and thereby determining the introduction of a nucleic acid into the genome of the mammalian cell if the transfected cells is viable in the presence of the toxin.