Sequential Nuclease and Donor Administration for Markerless Transgene Integration
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Solution Overview
Problem
Current methods for nuclease-mediated targeted integration of transgenes are inefficient due to factors like accessibility of chromosomal DNA and binding interaction quality, requiring selectable markers that leave additional genetic material in the genome, which is undesirable for many applications.
Innovation Solution
Sequential administration of nucleases and transgenes with a delay between administrations, such as 24 to 72 hours, to enhance the efficiency of targeted integration, using engineered nucleases like zinc finger nucleases, TALENs, or CRISPR/Cas systems, allowing for precise integration without the need for selectable markers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If selectable markers are used during donor integration to select modified variants, then the success rate of nuclease-driven genomic modifications increases, but additional genetic material is inserted into the genome which is undesirable for many applications
Solution Approach 1:
The patent removes the selectable marker from the integration process by using a markerless donor construct. Instead of relying on antibiotic resistance or other selectable markers to identify successfully modified cells, the method uses the nuclease-induced double-strand break itself as the selection mechanism, allowing only cells that have integrated the donor at the target site to survive or be identified through alternative means such as fluorescence-activated cell sorting (FACS) or PCR screening.
Solution Approach 2:
The patent performs preliminary chromosomal breakage by delivering the nuclease (e.g., zinc finger nuclease, TALEN, or CRISPR/Cas system) before introducing the donor DNA. This pre-created double-strand break ensures that when the markerless donor is introduced, it will be efficiently integrated via homology-directed repair or non-homologous end joining, eliminating the need for subsequent selection based on marker genes.
2Ease of operation
If nucleases and transgenes are administered concurrently or in rapid succession, then the process is simplified, but the efficiency of targeted integration is low due to factors like chromosomal DNA accessibility and binding interaction quality
Solution Approach 1:
The patent divides the gene modification process into distinct temporal phases: first delivering the nuclease to create chromosomal breaks and prepare the target site, then introducing the donor DNA at a later time point (e.g., 6-48 hours later). This segmentation allows each component to perform its function optimally without competition or interference, significantly improving integration efficiency compared to concurrent administration.
Solution Approach 2:
The nuclease is administered in advance to create double-strand breaks and recruit repair machinery to the target locus before the donor DNA is introduced. This preliminary action prepares the chromosomal landscape for efficient donor integration, ensuring that when the donor arrives, the cellular repair systems are already activated and positioned at the correct genomic location.
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 efficiency of transgene integration by 10 to 100-fold compared to concurrent or serial administration methods, minimizing gene silencing risks and avoiding unwanted genetic material insertion.
Implementation Method 1
Cleavage of a target nucleotide sequence by these nucleases increases the frequency of homologous recombination (HR) with a donor at the targeted locus by more than 1000-fold
Implementation Method 2
the repair of a site-specific DSB by non-homologous end joining (NHEJ) can also result in gene modification, including gene (transgene) insertion by NHEJ-dependent end capture
Data Source
AI summary
Disclosed herein are methods and compositions for targeted, nuclease-mediated insertion of transgene sequences into the genome of a cell.