DNA Editing Vector With Split Marker for Foreign-Gene-Free Cell Selection
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Solution Overview
Problem
Conventional DNA insertion methods are inefficient and result in the presence of foreign genes in genetically modified cells, limiting their usefulness, and existing DNA editing technologies lack a method for accurate and efficient insertion or removal of nucleotide sequences at target sites.
Innovation Solution
A DNA editing method using a Non-Integrative Cleavage-dependent system (NICS) that employs a vector with a divided selection marker gene and a site-specific nuclease to insert or remove nucleotide sequences at target sites, utilizing homologous sequences for precise binding and a CEN/ARS sequence for vector elimination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional DNA insertion methods using selection marker genes are used, then cell selection for confirming insertion is achieved, but foreign genes remain in the genetically modified cells reducing their usefulness
Solution Approach 1:
The selection marker gene is divided into two separate fragments that are introduced into the cell at different times. The first fragment is introduced with the target nucleotide sequence, and the second fragment is introduced later to complete the functional selection marker gene only in cells that have successfully integrated the target sequence, thereby eliminating the need for persistent foreign genes.
Solution Approach 2:
The first fragment of the selection marker gene is introduced together with the target nucleotide sequence to enable subsequent selection. This preliminary introduction allows for efficient cell selection while the complete functional gene is only formed after successful integration, minimizing foreign gene presence.
2Manufacturing precision
If homologous recombination is used for DNA insertion, then accurate insertion at target site is achieved, but the vector becomes very long and difficult to prepare
Solution Approach 1:
The homologous recombination vector is divided into two separate vectors. The first vector contains the target nucleotide sequence flanked by homologous sequences and a first fragment of the selection marker gene, while the second vector contains the remaining fragment. This segmentation reduces the complexity and length of each individual vector while maintaining accurate insertion capability through homologous recombination.
3Ease of manufacture
If microhomology-mediated end joining is used for DNA insertion, then vector preparation is simplified, but insertion efficiency and accuracy are reduced
Solution Approach 1:
The insertion system is segmented into two vectors introduced at different times. The first vector uses microhomology sequences for initial integration, simplifying vector preparation. The second vector containing the remaining selection marker gene fragment ensures accurate selection and verification of successful insertion, thereby maintaining precision despite the simplified approach.
4Reliability
If selection marker genes are always present in edited cells, then cell selection is reliable, but the utility of genetically modified cells is limited
Solution Approach 1:
The selection marker gene is segmented and temporarily present only during the selection phase. After successful integration of the target sequence, the complete functional selection marker gene enables reliable cell selection. Subsequently, the selection marker can be removed or inactivated, allowing the genetically modified cells to be used for various applications without the limitation of permanent foreign gene presence.
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 accurate and efficient insertion or removal of nucleotide sequences at target DNA sites, allowing for the production of cells without foreign genes, enhancing the utility of genetically modified cells.
Implementation Method 1
a method for specifically cleaving a target site of a target DNA in a cell or the vicinity thereof and editing the nucleotide sequence of the target site, using site-specific nuclease systems
Implementation Method 2
by replacing the desired nucleotide sequence with the target sequence or inserting the desired nucleotide sequence into the target sequence by means of homologous recombination (HR)
Data Source
Figure 1A(a)~1A(b)
Figure 1B(a)~1B(b)
Figure 1C(a)~1C(b)
AI summary
A method for editing DNA, which is a method for inserting a desired nucleotide sequence at a target site of a target DNA in a cell or removing the target site, the method comprising an introduction step of introducing a vector and a site-specific nuclease system into a cell to bring them into contact with a target DNA, wherein the vector comprises a first promoter P1 and structure (1): 5'-M1-Hv1-D-Hv2-M2-3', wherein M1 represents a 5' side fragment of a nucleotide sequence encoding a selection marker gene and is operably linked to P1, Hv1 represents a nucleotide sequence homologous to a first nucleotide sequence Ht1 on the 5' side of the target site of the target DNA, D represents the desired nucleotide sequence but may be absent, Hv2 represents a nucleotide sequence homologous to a second nucleotide sequence Ht2 on the 3' side of the target site of the target DNA, and M2 represents the remaining 3' side fragment of the nucleotide sequence encoding the selection marker gene, a cleavage step of generating from the vector, by the site-specific nuclease system, a fragment represented by structure (2): 5'-Hv1-D-Hv2-3' and a fragment represented by structure (3): 5'-M2-P1-M1-3', and cleaving the target site or its vicinity in the target DNA; an editing step in which the target DNA and structure (2) bind depending on the homology between Ht1 and Hv1 and bind depending on the homology between Ht2 and Hv2, and the desired nucleotide sequence D is inserted at the target site or the target site is removed; and a selection step of obtaining a selection vector in which, in the fragment represented by structure (3), the 3' end of M1 and the 5' end of M2 are ligated to obtain a selection vector that contains a functional selection marker gene operably linked to P1.