Yeast Genome Editing With Pre-Assembled RNPs for Multiplex Targeting
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Solution Overview
Problem
Current genome editing methods for yeast strains like Komagataella phaffii are inefficient, costly, and prone to off-target effects due to the use of DNA-based expression cassettes for Cas endonucleases and guide-RNAs, leading to low targeting efficiency and high construction costs for multiple gene modifications.
Innovation Solution
A method utilizing in vitro pre-assembled ribonucleoproteins (RNPs) and donor-DNA constructs with short homology arms for targeted genome editing, eliminating the need for selectable markers and reducing the size and complexity of DNA constructs, allowing simultaneous multiplex editing with reduced off-target activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If DNA-based expression cassettes are used for Cas endonucleases and guide-RNAs, then genome editing can be performed, but the construction cost increases and efficiency decreases
Solution Approach 1:
The patent divides the genome editing system into separate components: pre-assembled ribonucleoprotein complexes (Cas endonuclease + guide RNA) and donor DNA templates. This segmentation allows the RNP complex to be prepared and validated separately, then introduced into cells with minimal DNA construct complexity, reducing construction costs and improving efficiency.
Solution Approach 2:
The ribonucleoprotein complexes are pre-assembled in vitro before introduction into the cell. This preliminary action ensures proper complex formation and functionality outside the cell, eliminating the need for complex DNA expression cassettes that would require cellular transcription and translation machinery, thereby simplifying the overall system.
2Reliability
If DNA-based expression cassettes are used, then Cas endonucleases and guide-RNAs can be delivered, but off-target effects increase
Solution Approach 1:
The patent uses transient introduction of pre-assembled RNP complexes that perform their cutting function quickly before being degraded by cellular nucleases. This rapid action window reduces the time for off-target effects to occur, improving targeting accuracy while maintaining editing efficiency.
Solution Approach 2:
The RNP complexes are designed as transient, short-lived entities that are introduced, perform their function, and then degraded. This disposable nature ensures they do not persist in the cell to cause off-target effects, unlike stable DNA expression cassettes that continuously produce Cas endonuclease and guide RNA.
3Productivity
If selectable markers are used in genome editing, then edited cells can be selected, but the editing process becomes less efficient and more costly
Solution Approach 1:
The patent extracts the selection marker from the minimal donor DNA template, using only the essential homology arms and desired genetic modification. This extraction reduces the donor DNA size and complexity while maintaining the ability to select for successful editing events through alternative methods.
Solution Approach 2:
Instead of incorporating selectable markers into the donor DNA template, the patent inverts the approach by using the presence of the RNP complex itself and the specific genomic modification as the selection criterion, thereby simplifying the donor construct and increasing editing throughput.
4Adaptability or versatility
If multiple genes are modified using traditional methods, then genetic diversity is achieved, but construction cost and time increase significantly
Solution Approach 1:
The patent segments the multiplex editing strategy into separate RNP complexes, each targeting a specific gene. Multiple RNPs can be introduced simultaneously in a single transfection event, enabling parallel editing of multiple genes without the need to construct and test multiple separate DNA expression cassettes, thereby reducing time and cost.
Solution Approach 2:
The patent uses a universal RNP delivery platform that can accommodate multiple different guide RNAs targeting different genes. This multi-functional approach allows the same Cas endonuclease backbone to be paired with various guide RNAs for simultaneous delivery to cells, achieving multiplex editing with a single system rather than requiring gene-specific expression constructs for each target.
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 and cost-effectiveness of genome editing in yeast by enabling marker-less editing, reducing the risk of off-target mutations, and accelerating the process of genetic manipulation for protein expression.
Implementation Method 1
The programmable Cas9 enzyme has two RNA-guided DNA endonuclease domains capable of targeting specific genomic sequences
Implementation Method 2
wherein the donor-DNA construct serves as a template for the repair of the break by homologous recombination repair
Data Source
AI summary
The present invention relates to a method for editing the genome of a cell, such as a yeast cell. The method of the present invention requires the cell to be contacted with at least one ribonucleoprotein, at least one donor-DNA construct and a selectable marker such that they are introduced into the cell. The present invention is especially suitable for multiplex genome editing of cells such as yeast cells. The current invention further relates to a composition, a cell obtainable by the method of the invention and a method for the production of a compound of interest.