Split Selection Marker for Microorganism Gene Targeting
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Solution Overview
Problem
Efficient gene targeting in microorganisms like Saccharomyces pastorianus for lager brewing is challenging due to low homologous recombination efficiency, leading to increased false positives and the need for longer homologous sequences, which complicates genetic modifications and marker recycling.
Innovation Solution
A set of targeting constructs is used, splitting the selection marker into two parts with overlapping regions, each containing at least 20 bp of homology, allowing for enhanced recombination events and scarless marker removal through homologous recombination, thereby improving the ratio of true positives over false positives.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If longer homologous sequences are used to improve gene targeting efficiency in Saccharomyces pastorianus, then the percentage of correct integrations increases, but the complexity of genetic modifications and marker recycling increases
Solution Approach 1:
The selection marker is divided into two separate parts (first part and second part) that can be integrated independently into the genome. Each part contains homologous sequences for recombination, allowing the marker to be split across different genomic locations. This segmentation enables more flexible and efficient gene targeting without requiring single long homologous sequences, thereby reducing the complexity of genetic modifications while maintaining high integration accuracy.
2Productivity
If marker recycling is implemented for multiple gene deletions, then productivity increases, but the occurrence of chromosomal rearrangements increases due to residual sequences
Solution Approach 1:
The invention enables complete removal of the selection marker from the genome after it has served its purpose. By splitting the marker into two parts that can be independently integrated and then completely excised through recombination, the system allows marker recycling for multiple gene deletions without leaving residual sequences that could cause chromosomal rearrangements. This extraction of the marker after use eliminates the harmful effect of residual sequences while maintaining high productivity for multiple genetic modifications.
3Ease of operation
If conventional single marker integration is used, then the process is simple, but the ratio of true positives over false positives decreases
Solution Approach 1:
The selection marker is segmented into two distinct parts that are integrated at different genomic locations. This segmentation creates a more rigorous selection process where both parts must be correctly integrated for the organism to survive, significantly reducing false positives. The first part contains a first homologous sequence and the second part contains a second homologous sequence, requiring two independent recombination events for successful integration, thereby enhancing measurement precision while maintaining operational simplicity.
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 enhances the percentage of correctly integrated constructs and allows for seamless gene deletion and marker recycling, reducing the occurrence of chromosomal rearrangements and increasing the efficiency of genetic modifications in microorganisms.
Implementation Method 1
an exonuclease degrades both 5' ends
Implementation Method 2
Homologous recombination in microorganisms such as yeast is based on a double strand break repair mechanism, which joins the DNA fragments
Implementation Method 3
the heterodimer of so called Ku proteins grasps the broken chromosome ends, which promotes the binding of additional proteins
Implementation Method 4
The DNA synthesis mechanism repairs both strands
Implementation Method 5
DNA ligation completes the process without any deletions
Data Source
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AI summary
Title: Novel genome alteration system for microorganisms The invention relates to a set of targeting constructs, comprising a first construct comprising a recognition site for an endonuclease, a first region of homology with a target gene of a microorganism, and a first part of a selection marker, and a second construct comprising a second part of the selection marker, a second region of homology with the target gene of the microorganism, and a copy of the endonuclease recognition site. The invention further relates to methods for altering a target gene in a microorganism, to methods for producing a microorganism, and to microorganisms that are produced by the methods of the invention.