Guide RNA 5' End Tuning for Precise Transcription Control
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Solution Overview
Problem
There is a need for further technological development in controlling RNA transcription and applying RNA-guided nucleases, with a focus on designing expression vectors and kits for enhanced control over transcription and genome editing.
Innovation Solution
The method involves adding three or more nucleotide residues to the 5' end of guide RNA and adjusting the spacer sequence to control transcription and editing, utilizing RNA-guided nucleases like Cas proteins from Type V CRISPR/Cas systems, and incorporating fusion proteins with transcription regulation domains for targeted gene control.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If nucleotide residues are added to the 5' end of guide RNA to control transcription, then transcription control precision is improved, but guide RNA structure complexity increases
Solution Approach 1:
The patent applies parameter changes by modifying the 5' end of guide RNA with specific nucleotide residues (such as uracil, cytosine, guanine, or adenine) to control transcription. By changing the nucleotide composition and sequence at the 5' end, the invention achieves precise control over transcription without fundamentally altering the overall guide RNA structure, thus resolving the contradiction between control precision and structural complexity.
2Productivity
If spacer sequence is adjusted to enhance editing efficiency, then genome editing efficiency is improved, but off-target effects increase
Solution Approach 1:
The patent applies local quality by carefully designing and adjusting specific regions of the spacer sequence while maintaining the overall functionality of the guide RNA. By optimizing local nucleotide compositions and sequences in the spacer region, the invention enhances editing efficiency at the target site while minimizing off-target effects through localized sequence-specific binding, thus resolving the contradiction between editing efficiency and off-target effects.
3Adaptability or versatility
If RNA-guided nucleases are used for transcription control, then transcription regulation capability is improved, but system complexity increases
Solution Approach 1:
The patent applies universality by utilizing RNA-guided nucleases that can perform multiple functions including transcription control, genome editing, and base editing. By designing a versatile system where the same core components (guide RNA with modified 5' end and RNA-guided nuclease) can be applied to different transcription regulation scenarios, the invention enhances transcription regulation capability while avoiding the need for entirely separate systems for each application, thus reducing overall system complexity.
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 precise control over RNA transcription and genome editing, enhancing the efficiency and specificity of RNA-guided nuclease applications, including base editing and transcription regulation.
Implementation Method 1
The Cas9-crRNA-tracrRNA complex binds to foreign invasive DNA complementary to the crRNA
Implementation Method 2
a step of transcribing RNA from an expression vector for the RNA
Implementation Method 3
the RNA is RNA to which one or more nucleotide residues are added at the 5' end
Data Source
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AI summary
Provided is a method for controlling transcription of RNA, including: a step of transcribing RNA from an expression vector for the RNA, in which the RNA is RNA to which one or more nucleotide residues are added at the 5' end, and the like. In addition, provided is a method for designing an expression vector for RNA, including: designing an expression vector for RNA in which transcription efficiency of the RNA is controlled by adjusting one or more selected from the group consisting of the number of nucleotide residues to be added to the 5' end of an RNA coding sequence and the type of the nucleotide residues, and the like.