Guide-RNA Expression System Using Viral Polymerases
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Solution Overview
Problem
Current guide-RNA expression systems for RNA-guided nuclease systems, particularly in fungal cells like yeast and filamentous fungi, are complex, require bulky expression cassettes, and lack straightforward tuning, limiting their application in genome editing and regulation due to the transient nature of in vitro transcribed guide-RNAs.
Innovation Solution
The use of single-subunit DNA-dependent RNA polymerases such as T3, SP6, K11, or T7 RNA polymerases to express guide-RNAs within fungal cells, where the guide-RNA is encoded by a polynucleotide operably linked to viral single-subunit RNA polymerase promoters, enabling efficient and tunable expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Duration of action of moving object
If in vitro transcribed guide-RNAs are introduced into host cells, then guide-RNA expression can be achieved, but the transient nature limits the duration and stability of guide-RNA availability
Solution Approach 1:
The system divides the guide-RNA expression function into two separate components: a DNA template containing the guide-RNA sequence and a viral RNA polymerase enzyme. This segmentation allows the DNA template to be stably maintained in the host cell genome while the viral polymerase provides transient expression capability, resolving the contradiction between duration and reliability.
Solution Approach 2:
The viral single-subunit RNA polymerase acts as an intermediary that bridges the stable DNA template and the need for guide-RNA production. The polymerase recognizes viral promoters on the DNA template and catalyzes guide-RNA synthesis, enabling stable yet controllable expression without requiring the host cell's complex eukaryotic transcription machinery.
2Productivity
If complex expression cassettes are used for guide-RNA expression in fungal cells, then guide-RNA can be produced, but the device complexity increases and tuning capability is limited
Solution Approach 1:
The invention extracts the essential transcription function from the complex eukaryotic RNA polymerase II system and isolates only the critical viral RNA polymerase component. This extraction eliminates the need for bulky expression cassettes containing multiple eukaryotic regulatory elements, achieving high productivity with minimal complexity.
Solution Approach 2:
The system enables straightforward tuning of guide-RNA expression by changing parameters such as promoter strength, polymerase concentration, and guide-RNA sequence. These parameter changes provide precise control over expression levels without requiring complex cassette redesign, maintaining both productivity and simplicity.
3Ease of manufacture
If eukaryotic RNA polymerase III promoters are used for guide-RNA expression, then guide-RNA can be generated without 5' cap, but the expression system becomes bulky and less tunable
Solution Approach 1:
The viral RNA polymerase system functions as a disposable, transient expression mechanism. The polymerase is introduced temporarily to synthesize guide-RNA from the DNA template, then its function is complete. This approach achieves ease of manufacture without requiring permanent integration of complex eukaryotic promoter elements into the host genome.
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 guide-RNA expression in fungal cells, enhancing the stability and availability of guide-RNAs for RNA-guided nuclease systems, thereby improving genome editing and regulation capabilities.
Implementation Method 1
transcription of the guide-RNA is performed by a viral single-subunit DNA-dependent RNA polymerase
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 guide-RNA expression system for a eukaryotic host cell.