Oligonucleotide Library for High-Throughput Yeast Metabolic Engineering
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Solution Overview
Problem
Current methods for metabolic engineering and genetic manipulation are inefficient and time-consuming due to limited selection markers and the need for successive rounds of screening to introduce or modify genes in organisms, hindering the production of chemicals, fuels, and medicines.
Innovation Solution
A method involving the synthesis of oligonucleotides encoding guide nucleic acids and donor DNA sequences, which are incorporated into plasmids and expressed in yeast cells to introduce targeted mutations, enabling the creation of libraries with comprehensive diversity and efficient recombination at specific sites, allowing for high-throughput genetic modification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If standard gene targeting methods using PCR-generated marker cassettes are used, then genes can be introduced or removed from organisms, but the process is time-consuming and inefficient due to limited selection markers requiring successive rounds of screening
Solution Approach 1:
The invention segments the gene targeting process into two independent components: a plasmid backbone containing selection markers and a separate oligonucleotide containing the guide nucleic acid and donor DNA. This allows simultaneous introduction of multiple genes in a single transformation event, eliminating successive screening rounds and dramatically improving productivity.
Solution Approach 2:
The invention performs preliminary action by pre-synthesizing oligonucleotides with specific guide nucleic acids and donor DNA sequences before transformation. This pre-prepared oligonucleotide library enables parallel introduction of multiple genes without requiring time-consuming successive screening rounds, as all targeting information is already encoded in the oligonucleotides.
2Adaptability or versatility
If limited selection markers are used, then the number of genes that can be removed and heterologous genes inserted is limited, but expanding selection markers would require complex additional systems
Solution Approach 1:
The invention applies universality by using a single plasmid backbone that can accommodate multiple different oligonucleotides with various guide nucleic acids and donor DNA sequences. This universal plasmid system enables modification of any gene in the host organism by simply changing the oligonucleotide, without requiring different selection marker systems for each gene targeting application.
Solution Approach 2:
The oligonucleotide serves as an intermediary carrier that delivers both the guide nucleic acid and donor DNA to the host cell. This intermediary molecule enables versatile gene modification by carrying different targeting information without requiring the host cell to have multiple specialized selection marker systems, thus increasing adaptability without proportionally increasing complexity.
3Productivity
If conventional gene targeting methods are used, then genetic modification can be achieved, but the process lacks high-throughput capability and cannot generate comprehensive mutant libraries efficiently
Solution Approach 1:
The invention performs preliminary action by synthesizing a library of oligonucleotides with different guide nucleic acids and donor DNA sequences before transformation. This pre-assembled oligonucleotide library can be introduced into host cells in a single high-throughput transformation event, generating comprehensive mutant libraries without the time loss associated with conventional step-by-step gene targeting methods.
Solution Approach 2:
The invention merges the plasmid backbone with the oligonucleotide containing guide nucleic acid and donor DNA into a single transformation event. This merging of components enables simultaneous introduction of multiple genes and creation of comprehensive mutant libraries in parallel, dramatically increasing throughput and reducing the time required for library generation compared to sequential conventional methods.
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 enables rapid and efficient generation of mutant yeast cells with diverse genetic modifications, significantly reducing the time and cost associated with metabolic engineering and allowing for the optimization of metabolic pathways and production of desired compounds.
Implementation Method 1
The guide nucleic acid is complementary to a target site in a nucleic acid in a yeast cell
Implementation Method 2
The nucleic acid-guided endonuclease breaks the nucleic acid at the target site
Implementation Method 3
The donor DNA recombines with the nucleic acid at the two regions via the two sequences which are complementary, rejoining two portions of the nucleic acid that had been separated by breakage
Data Source
AI summary
Expressing guide nucleic acids (e.g., gRNA) from the same oligonucleotide that contains donor sequence permits the high efficiency, simultaneous transformation of a population of cells with both substrates. Using oligonucleotide chip array technology, one can construct thousands of oligonucleotides with customized gRNA and donor sequence in a cost effective manner. In combination, one can efficiently modify endogenous and exogenous genes.