Targeted Genomic Alteration via Multiple Insertion Sites
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for targeted genome modification in plants and mammalian cells are inefficient and non-specific, leading to unpredictable outcomes and pleiotropic effects due to random integration of exogenous DNA, which complicates the generation and characterization of transgenic lines with desired traits.
Innovation Solution
The use of multiple insertion sites with specific target sites for zinc finger nucleases (ZFNs) allows for targeted integration and expression of exogenous nucleic acid sequences, enabling controlled and specific integration of genes into predetermined locations within the genome through homology-dependent and homology-independent mechanisms.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional plant transformation methods are used to deliver exogenous DNA, then transgenic lines can be generated, but the integration occurs at random locations requiring screening of thousands of unique random-integration events
Solution Approach 1:
The patent introduces a multiple insertion site (MIS) construct that is pre-integrated into the plant genome at a specific locus before the exogenous gene of interest is delivered. This preliminary placement creates a predetermined target location, eliminating the need to screen thousands of random integration events and directly enabling targeted integration of the transgene at the pre-established MIS location.
2Productivity
If exogenous DNA integrates into the genome at random locations, then transgenic lines can be obtained, but unintended genome disruption and pleiotropic effects occur
Solution Approach 1:
The multiple insertion site (MIS) construct serves as an intermediary element between the exogenous gene of interest and the plant genome. The MIS contains specific recognition sequences for homing endonucleases and provides a safe harbor location with necessary regulatory elements, mediating the integration process to ensure the transgene is inserted at a predetermined safe location without disrupting essential genomic functions.
3Manufacturing precision
If targeted gene modification is attempted in plant species, then specific locus modification is achieved, but the process remains difficult and inefficient without pre-engineered restriction sites
Solution Approach 1:
The patent divides the targeted integration system into separate functional modules: (1) a pre-integrated multiple insertion site construct containing homing endonuclease recognition sequences and regulatory elements, (2) the exogenous gene of interest delivered separately, and (3) homing endonucleases that recognize specific sequences. This segmentation allows each component to be optimized independently and simplifies the overall process by separating the targeting function from the transgene delivery.
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 approach facilitates stable, heritable genetic modifications in plants and mammalian cells, reducing the risk of unintended genome disruption and improving the efficiency and predictability of transgenic trait development.
Implementation Method 1
The use of multiple insertion sites with specific target sites for zinc finger nucleases (ZFNs) allows for targeted integration and expression of exogenous nucleic acid sequences
Implementation Method 2
enabling controlled and specific integration of genes into predetermined locations within the genome through homology-dependent and homology-independent mechanisms
Data Source
AI summary
Disclosed herein are methods and compositions for targeted integration and/or targeted excision of one or more sequences into a cell, for example, for expression of one or more polypeptides of interest.