Multi-Vector Homologous Recombination for Large Genomic Replacement
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Solution Overview
Problem
Existing methods for genome modification using homologous recombination face challenges with low targeting frequency, particularly with large targeting vectors and specific cell types such as fibroblasts, when replacing large genomic fragments like human sequences in rodent genomes.
Innovation Solution
A method involving the use of two or more large targeting vectors (LTVECs) that recombine to form a single contiguous nucleic acid segment, facilitated by a nuclease agent to introduce targeted breaks, and integration of nucleic acid inserts flanked by homology arms, enhancing targeting efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Volume of moving object
If large targeting vectors (LTVECs) are used to replace large genomic fragments, then the size of the modified genomic region increases, but the targeting frequency decreases
Solution Approach 1:
The patent divides a large targeting vector into multiple smaller LTVECs (first LTVEC, second LTVEC, and further LTVECs) that can be introduced separately into the cell. These segmented vectors each contain smaller nucleic acid inserts flanked by homology arms, making them more efficient at targeting the genomic locus while collectively achieving the replacement of large genomic fragments through their combined integration.
2Adaptability or versatility
If very large targeting vectors are used to target specific cell types such as fibroblasts, then the capability to modify complex genomic regions improves, but the targeting efficiency deteriorates
Solution Approach 1:
The patent segments very large targeting vectors into multiple smaller LTVECs that can be more efficiently taken up and recombined in specific cell types like fibroblasts. Each LTVEC contains a manageable nucleic acid insert with homology arms, improving cellular uptake and recombination efficiency while maintaining the capability to modify complex genomic regions through the combined action of multiple vectors.
Solution Approach 2:
The patent combines multiple smaller LTVECs (first LTVEC, second LTVEC, and further LTVECs) to achieve the cumulative effect of a very large targeting vector. The vectors work together through homologous recombination to replace large genomic fragments, merging their individual targeting capabilities to achieve both high efficiency and complex genomic modification capability.
3Reliability
If multiple large targeting vectors are introduced to achieve high targeting efficiency, then the targeting frequency improves, but the complexity of the system increases
Solution Approach 1:
The patent segments the targeting system into multiple modular LTVECs, where each vector is a discrete, manageable unit with defined homology arms and nucleic acid inserts. This segmentation allows for systematic combination of vectors to achieve desired targeting frequency while maintaining control over system complexity through modular design.
Solution Approach 2:
The patent creates universal LTVEC components with standardized homology arms and modular nucleic acid inserts that can be combined in various configurations. The first LTVEC, second LTVEC, and further LTVECs share common structural features and recombination mechanisms, allowing the system to achieve multiple targeting outcomes through a unified, multi-functional vector platform that reduces overall system complexity.
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 targeting efficiency, allowing for precise genomic modifications, including additions, deletions, and swaps, with up to 20-fold improvement in some cases, and enables the production of modified non-human animals.
Implementation Method 1
Homologous recombination using targeting vectors designed to add, delete, or replace a particular nucleic acid sequence at a genomic locus
Data Source
AI summary
Methods and compositions are provided for making one or more targeted genetic modifications at a target genomic locus within a cell and for producing non-human animals comprising the modified genomic locus. The methods employ two or more large targeting vectors which are capable of recombining with each other and with the target genomic locus in a single genomic targeting step. The methods may also be employed in combination with a nuclease agent. Methods and compositions are also provided for enhancing homologous recombination at a target genomic locus in a cell. The methods employ two or more nucleic acids comprising one or more overlapping sequences. The methods may be employed in combination with a nuclease agent or without a nuclease agent.