tRNA Promoter Guide RNA Expression in Non-Conventional Yeast
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Solution Overview
Problem
Current genome engineering technologies, such as CRISPR systems, face challenges in specificity, cost-effectiveness, and scalability, particularly when targeting multiple genomic positions, and are inefficient in certain organisms, requiring complex and labor-intensive procedures.
Innovation Solution
A recombinant DNA construct comprising a tRNA promoter linked to a polynucleotide encoding a single guide RNA, without a ribozyme, forms a guide RNA/Cas endonuclease complex that binds and cleaves target sites in non-conventional yeast, enhancing mutation efficiency up to 20-fold compared to ribozyme-linked systems.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR systems are used for genome engineering, then targeted DNA strand breaks can be introduced, but the systems have low specificity and require redesign for each target site which is costly and time-consuming
Solution Approach 1:
The patent employs a universal Cas9 endonuclease that can target multiple genomic positions when combined with different guide RNAs. The expression construct uses a tRNA promoter that can drive expression of multiple sgRNAs simultaneously, allowing a single system to perform multiple targeting functions without redesigning the core machinery for each target site.
Solution Approach 2:
The patent segments the guide RNA into multiple individual sgRNA expressions that are processed from a single transcript. The tRNA promoter system allows independent expression units (each sgRNA separated by tRNA sequences) to be transcribed as one unit but function independently, enabling modular targeting of multiple sites simultaneously.
2Ease of manufacture
If traditional expression constructs with ribozyme are used, then guide RNA can be expressed, but the system is complex and labor-intensive
Solution Approach 1:
The patent removes the ribozyme component from the expression construct while maintaining guide RNA expression functionality. By eliminating the ribozyme self-cleavage mechanism and relying instead on tRNA promoter-driven expression followed by cellular processing, the system becomes simpler without requiring additional enzymatic activities or complex secondary structures.
Solution Approach 2:
The expression construct leverages the cell's own tRNA processing machinery to handle the guide RNA expression and maturation. The tRNA promoter is recognized by cellular RNA polymerase, and the resulting transcript is processed by cellular nucleases and modification enzymes, allowing the system to utilize existing cellular infrastructure rather than requiring additional engineered components.
3Manufacturing precision
If homologous recombination is used for gene knock-out, then targeted insertions can be achieved, but the procedure is complex and labor-intensive
Solution Approach 1:
The patent replaces the mechanical process of homologous recombination with a biochemical cleavage system. Instead of relying on cellular recombination machinery to achieve targeted modifications, the Cas9-sgRNA complex directly introduces double-strand breaks at specific locations, which then trigger simplified repair pathways that can be harnessed for knock-out or knock-in without requiring complex homologous recombination protocols.
4Productivity
If CRISPR systems are designed to target multiple positions, then scalability is improved, but specificity and efficiency decrease in non-conventional yeast
Solution Approach 1:
The patent optimizes the expression parameters by using tRNA promoters that are specifically adapted for non-conventional yeast. The tRNA promoter system allows for controlled expression levels of multiple sgRNAs, and the use of different tRNA genes (with varying expression strengths) enables tuning of the relative abundance of each guide RNA to match the requirements for multiplexed targeting in yeast cells.
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 provides a more efficient, affordable, and scalable method for editing nucleotides and altering target sites in yeast genomes, improving the precision and throughput of genome engineering.
Implementation Method 1
a tRNA promoter operably linked to a polynucleotide encoding a single guide RNA
Implementation Method 2
said guide RNA is capable of forming a guide RNA/Cas endonuclease complex
Implementation Method 3
said complex can bind to and cleave a target site sequence in the genome of a non-conventional yeast
Data Source
Figure 1
Figure 2
Figure 3A~3B
AI summary
Compositions and methods are provided for editing nucleotides and/or altering target sites in the genome of a cell. The methods and compositions employ a recombinant DNA construct comprising a tRNA promoter operably linked to a polynucleotide encoding a single guide RNA, wherein said recombinant DNA construct does not comprise a nucleotide sequence encoding a ribozyme, wherein said guide RNA is capable of forming a guide RNA/Cas endonuclease complex, wherein said complex can bind to and cleave a target site sequence in the genome of a cell such as a microbial cell. The present disclosure further describes methods and compositions employing a recombinant DNA construct comprising a tRNA promoter operably linked to a spacer sequence and a polynucleotide encoding a single guide RNA, wherein said recombinant DNA construct does not comprise a nucleotide sequence encoding a ribozyme, wherein said guide RNA is capable of forming a guide RNA/Cas endonuclease complex, wherein said complex can bind to and cleave a target site sequence in the genome of a non-conventional yeast.