Non-disruptive Gene Targeting via 2A Peptide Cassette
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Solution Overview
Problem
Current gene targeting technologies often disrupt the expression of genes at the target locus, leading to unwanted effects when integrating a gene of interest, which is undesirable for medical and biotechnological applications.
Innovation Solution
A donor polynucleotide composition comprising a nucleic acid cassette with the gene of interest, a 2A peptide, and sequences homologous to the target locus, allowing for targeted integration without disrupting the expression of the gene at the target locus, using techniques like homologous recombination and targeted nucleases.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If gene targeting is performed to integrate a gene of interest into a target locus, then the gene of interest can be expressed in a spatially and temporally restricted pattern, but the expression of the gene at the target locus is disrupted
Solution Approach 1:
The donor polynucleotide is divided into distinct functional segments: homology arms for targeted integration, a 2A peptide sequence for protein cleavage, and the gene of interest. This segmentation allows the gene of interest to be integrated while preserving the endogenous gene's reading frame and expression capability through the self-cleaving 2A peptide mechanism.
Solution Approach 2:
The 2A peptide acts as an intermediary element between the gene of interest and the endogenous gene. It enables the integration of the gene of interest while maintaining the functional integrity of the endogenous gene by facilitating protein cleavage that restores the original gene product's functionality.
2Productivity
If gene integration is performed using conventional methods, then the gene of interest can be expressed, but unwanted effects occur due to disruption of the target locus gene expression
Solution Approach 1:
The donor polynucleotide incorporates a 2A peptide sequence that preemptively prevents the harmful effect of gene disruption by enabling self-cleavage. This preliminary design ensures that when integration occurs, the endogenous gene's expression is automatically restored, preventing unwanted effects before they can manifest.
Solution Approach 2:
The invention changes the molecular parameters of the integrated construct by incorporating the 2A peptide sequence with specific self-cleaving properties. This parameter change transforms the integration outcome from gene disruption to gene preservation, allowing productive expression of the gene of interest without harmful side effects.
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 the expression of the gene of interest in a spatially and temporally restricted pattern while maintaining the normal expression of the gene at the target locus, suitable for various applications including gene therapy and production of genetically modified organisms.
Implementation Method 1
A donor polynucleotide composition comprising a nucleic acid cassette with the gene of interest, a 2A peptide, and sequences homologous to the target locus, allowing for targeted integration without disrupting the expression of the gene at the target locus, using techniques like homologous recombination
Data Source
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AI summary
Compositions and methods are provided for integrating one or more genes of interest into cellular DNA without substantially disrupting the expression of the gene at the locus of integration, i.e., the target locus. These compositions and methods are useful in any in vitro or in vivo application in which it is desirable to express a gene of interest in the same spatially and temporally restricted pattern as that of a gene at a target locus while maintaining the expression of the gene at the target locus, for example, to treat disease, in the production of genetically modified organisms in agriculture, in the large scale production of proteins by cells for therapeutic, diagnostic, or research purposes, in the induction of iPS cells for therapeutic, diagnostic, or research purposes, in biological research, etc. Reagents, devices and kits thereof that find use in practicing the subject methods are also provided.