CRISPR-Cas Yeast Genome Engineering via Guide Polynucleotides
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Solution Overview
Problem
Current genome engineering techniques, such as designer zinc fingers and TALENs, are challenging due to intertwined DNA recognition and cleavage functions, and constructing engineered zinc finger arrays is difficult, necessitating a more robust and affordable method for targeting specific sequences in host cells for various applications.
Innovation Solution
The CRISPR-Cas system, which uses a single Cas enzyme programmed by distinct guide-polynucleotides to recognize specific polynucleotide targets, simplifying genome engineering by not requiring customized proteins and facilitating targeted genome perturbations in yeast host cells like Saccharomyces and Kluyveromyces.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If designer zinc fingers or TALENs are used for genome engineering, then targeted genome perturbations can be achieved, but the device complexity and difficulty of construction increase significantly
Solution Approach 1:
The CRISPR-Cas system divides the genome editing function into separate components: a Cas enzyme for cleavage and guide-polynucleotides for targeting. This segmentation allows independent optimization of each component and simplifies construction compared to integrated systems like zinc fingers where recognition and cleavage are intertwined.
Solution Approach 2:
A single Cas enzyme can be programmed by different guide-polynucleotides to target multiple positions within a genome. This universal targeting capability eliminates the need to construct different protein arrays for each target site, significantly reducing construction complexity while maintaining reliable targeted perturbation.
2Measurement precision
If engineered zinc finger arrays are constructed, then specific sequences can be targeted, but the ease of manufacture decreases due to context-dependent effects
Solution Approach 1:
The targeting function is separated from the cleavage function. Guide-polynucleotides handle sequence recognition with simple base-pairing rules, while the Cas enzyme performs cleavage. This eliminates context-dependent effects between finger domains that complicate zinc finger array construction.
Solution Approach 2:
The system changes the molecular parameters of targeting from protein-DNA interactions (zinc fingers) to nucleic acid hybridization (guide-polynucleotide to target). Nucleic acid base-pairing follows simple, predictable rules without context-dependent effects, greatly simplifying the design and construction process while maintaining precise targeting.
3Measurement precision
If customized proteins are generated for each target, then specific polynucleotide sequences can be recognized, but the productivity and scalability decrease
Solution Approach 1:
A single Cas enzyme serves as a universal platform that can be programmed by different guide-polynucleotides to recognize and process multiple different target sequences. This eliminates the need to generate customized proteins for each target, dramatically increasing productivity and enabling high-throughput genome engineering applications.
Solution Approach 2:
Instead of creating new customized proteins for each target, the system uses identical Cas enzyme copies that are directed to different targets by synthesizing new guide-polynucleotides. Nucleic acid synthesis is much faster and more scalable than protein engineering, enabling rapid iteration and high productivity.
4Ease of operation
If a robust technique for targeting multiple positions is developed, then the ease of operation improves, but the device complexity may increase
Solution Approach 1:
The CRISPR-Cas system provides a universal platform where a single Cas enzyme can target multiple positions by simply changing the guide-polynucleotide sequence. This greatly improves ease of operation compared to systems requiring different proteins for each target, while the actual system complexity remains manageable due to the simplicity of nucleic acid-based programming.
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 simplifies genome engineering by allowing precise targeting and modulation of polynucleotides in yeast host cells, enhancing the scalability and ease of use of genome editing technologies, making them more accessible and robust for diverse applications.
Implementation Method 1
the guide-polynucleotides comprise a guide-sequence that essentially is the reverse complement of a target-polynucleotide in a host cell and the guide-polynucleotides can direct binding of the Cas protein at the target-polynucleotide in the host cell to form a CRISPR-Cas complex
Data Source
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AI summary
The present invention relates to the field of molecular biology and cell biology. More specifically, the present invention relates to a CRISPR-CAS system for a yeast host cell.