Target DNA Sequence Exchange via Direct Repeat Marker Excision

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

Existing methods for exchanging a target DNA sequence with a DNA sequence of interest in eukaryotic cells, such as plants, often require modifying the preselected site in the repair DNA, which can lower efficiency and introduce additional sequence variations.

Innovation Solution

A method involving inducing a double-stranded DNA break at a preselected site, introducing a repair DNA molecule with a selectable marker flanked by direct repeat sequences that are no longer recognized by the double-stranded break inducing enzyme, allowing for homologous recombination and subsequent removal of the marker without altering the preselected site.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If the preselected site in the repair DNA is modified to prevent recognition by the double-stranded break inducing enzyme, then the efficiency of DNA insertion by homologous recombination is improved, but additional sequence variations are introduced

Engineering Contradiction:

Improveefficiency of DNA insertionVSAvoidsequence accuracy

Core Design Contradiction:

ProductivityVSManufacturing precision

Solution Approach 1:

The recognition site is divided into two direct repeat sequences that flank the selectable marker. These repeats are recognized and recombined by the double-stranded break inducing enzyme, allowing precise excision of the marker while preserving the original recognition site sequence in the final construct.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The selectable marker flanked by direct repeats serves as an intermediary element. It is temporarily introduced to enable selection of homologous recombination events, then precisely removed through enzyme-mediated recombination of the repeats, leaving no footprint and restoring the original sequence.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Reliability

If a selectable marker is introduced into the repair DNA for temporal selection, then the selection of gene replacement events is improved, but the complexity of the DNA construct is increased

Engineering Contradiction:

Improveselection of gene replacement eventsVSAvoidDNA construct complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The selectable marker is introduced as a temporary element for selection purposes, then precisely removed through homologous recombination between the direct repeat sequences. This allows reliable selection of gene replacement events while ensuring the marker does not remain in the final construct, thus recovering the simplicity of the original sequence.

Inventive Principle:

Principle #34Discarding and recovering

3Productivity

If the preselected site is altered in the repair DNA to avoid cleavage, then the efficiency of homologous recombination is improved, but the original nucleotide sequence is lost

Engineering Contradiction:

Improveefficiency of homologous recombinationVSAvoidoriginal nucleotide sequence

Core Design Contradiction:

ProductivityVSStability of the object's composition

Solution Approach 1:

The direct repeat sequences are designed to be recognized by the double-stranded break inducing enzyme before the homologous recombination event. This preliminary arrangement allows the enzyme to create breaks at these sites, facilitating the excision of the marker and restoration of the original sequence after recombination.

Inventive Principle:

Principle #10Preliminary action

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 enhances the efficiency of DNA insertion by homologous recombination while maintaining the original nucleotide sequence of the preselected site, avoiding additional sequence modifications.

Implementation Method 1

Homologous recombination allows numerous targeted genetic modifications in prokaryotic and selected eukaryotic organisms

Methodology Applied

Scientific EffectHomologous recombination:

Implementation Method 2

induction of double stranded DNA breaks via rare-cutting endonucleases, such as e.g. I-SceI

Methodology Applied

Scientific EffectDouble stranded DNA break:

Data Source

PatentEP2160467B1Methods and means for exact replacement of target DNA in eukaryotic organisms

Publication Date: 2015.10.28 BAYER CROPSCIENCE NV

Figure 1A
Figure 1B
Figure 2A

AI summary

Methods and means are provided for the exact exchange in eukaryotic cells, such as plant cells, of a target DNA sequence for a DNA sequence of interest through homologous recombination, whereby the selectable or screenable marker used during the homologous recombination phase for temporal selection of the gene replacement events can subsequently be removed without leaving a foot-print employing a method for the removal of a selected DNA flanked by two nucleotide sequences in direct repeats.