Nuclease-Mediated Targeting with Large Vectors
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Solution Overview
Problem
Current methods face challenges in achieving efficient genomic modifications, particularly with large targeting vectors (LTVECs), especially when targeting large genomic loci or specific cell types like fibroblasts, due to low targeting frequency and difficulty in introducing human genomic fragments into non-human animal genomes.
Innovation Solution
The use of large targeting vectors (LTVECs) in combination with engineered nucleases that create single or double-strand breaks at target genomic loci, facilitated by recombinogenic prokaryotic cells like E. coli, to enhance homologous recombination efficiency, allowing for efficient deletion, addition, or replacement of nucleic acid sequences in mammalian and prokaryotic cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If large targeting vectors (LTVECs) are used to replace large genomic loci with human genomic fragments, then the ability to modify large genomic regions is improved, but the targeting frequency remains low
Solution Approach 1:
The patent divides the large targeting vector system into two separate components: (1) a small targeting vector containing homology arms and selection markers, and (2) a separate expression vector encoding the nuclease (e.g., ZFN or TALEN). This segmentation allows the small targeting vector to efficiently integrate into the genome at the nuclease-induced break site, while the nuclease handles the challenge of targeting large genomic regions. The separation resolves the contradiction by enabling high targeting frequency through the nuclease while maintaining the capability to modify large genomic loci through the homology arms of the small targeting vector.
Solution Approach 2:
The patent introduces an engineered nuclease (zinc-finger nuclease or TALEN) as an intermediary that creates double-strand breaks at specific genomic locations. This intermediary facilitates homologous recombination between the targeting vector and the genomic DNA, dramatically increasing targeting frequency. The nuclease acts as a mediator that overcomes the low efficiency inherent in using large targeting vectors alone, while still enabling replacement of large genomic fragments through the homology-directed repair pathway.
2Ease of operation
If conventional targeting vectors are used in fibroblasts or somatic cells, then the approach is simple, but the targeting efficiency is insufficient
Solution Approach 1:
The patent segments the targeting system into a small, easy-to-manipulate targeting vector and a separate nuclease expression system. This segmentation maintains operational simplicity because the small targeting vector can be easily constructed and introduced into cells, while the nuclease component can be delivered separately. The division allows each component to be optimized independently, resolving the contradiction between ease of operation and targeting efficiency in difficult-to-transfect cells like fibroblasts.
Solution Approach 2:
The patent changes key parameters of the targeting system by reducing the size of the targeting vector (from conventional large vectors to small vectors with minimal homology arms) and introducing nuclease activity as a new parameter. These parameter changes enable efficient targeting in somatic cells and fibroblasts, where conventional large vectors fail, while maintaining relative simplicity through the use of standard molecular biology techniques for vector construction and delivery.
3Quantity of substance
If large targeting vectors are used to achieve homologous recombination, then large genomic loci can be modified, but the process becomes complex and less efficient
Solution Approach 1:
The patent segments the complex large targeting vector into a simple small targeting vector and a separate nuclease expression vector. The small targeting vector contains only the essential elements (homology arms and selection marker), dramatically reducing its complexity. The nuclease expression vector handles the complexity of directing specific genomic modifications. This segmentation resolves the contradiction by enabling large genomic modifications through coordinated action of two simpler, less complex vector components.
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 significantly increases the targeting efficiency of LTVECs, achieving at least two-fold to four-fold higher targeting frequencies compared to using LTVECs alone, enabling precise genetic modifications in both prokaryotic and eukaryotic cells, including pluripotent mammalian cells and non-human animals.
Implementation Method 1
Homologous recombination using targeting vectors that are specifically designed to add, delete, or replace a particular nucleic acid sequence at a genomic locus
Data Source
AI summary
Compositions and methods are provided for making one or more targeted genetic modifications at a target genomic locus by employing homologous recombination facilitated by single or double-strand break at or near the target genomic locus. Compositions and methods for promoting efficiency of homologous recombination between an LTVEC and a target genomic locus in prokaryotic or eukaryotic cells using engineered nucleases are also provided.