CRISPR/Cas9 Activation of Endogenous RTP1 for Odorant Receptor Screening
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Solution Overview
Problem
Current methods for developing stable cell lines that express the endogenous RTP1 gene for improved odorant receptor function in non-olfactory cells are inefficient and cumbersome, lacking a straightforward technique for consistent expression without recombinant methods.
Innovation Solution
The use of CRISPR/Cas9 genome editing to introduce a guide RNA and Cas nuclease protein complex to activate the endogenous RTP1 gene, enabling the expression of the RTP1 protein, specifically targeting the genomic locus and using a constitutive promoter like CMV to drive expression, thereby enhancing odorant receptor activity in non-olfactory cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional molecular biology approaches are used to insert DNA into cultured cells to stably express the endogenous RTP1 gene, then the cell line can express RTP1 protein, but the process is inefficient and cumbersome
Solution Approach 1:
The patent replaces conventional mechanical DNA insertion methods (such as microinjection, electroporation, or viral transduction) with CRISPR/Cas9 genome editing. The CRISPR system uses guide RNA to direct the Cas9 nuclease to a specific genomic locus upstream of the RTP1 gene, creating a precise double-strand break that triggers endogenous repair mechanisms to integrate the constitutive promoter and achieve stable expression. This molecular precision eliminates the need for cumbersome screening and selection processes inherent in conventional methods.
2Reliability
If recombinant methods are used to develop stable cell lines expressing endogenous RTP1, then consistent protein expression can be achieved, but the techniques are cumbersome and complex
Solution Approach 1:
The patent employs the cell's own endogenous repair mechanisms (homology-directed repair and non-homologous end joining) to achieve gene activation. By providing a donor DNA template with homology arms flanking the constitutive promoter, the cell automatically integrates the promoter into the RTP1 locus through its natural repair pathways. This eliminates the need for complex recombinant DNA technologies, viral vectors, or extensive molecular biology expertise, simplifying the overall process while maintaining consistent protein expression.
3Productivity
If the endogenous RTP1 gene is activated using CRISPR/Cas9, then efficient and stable expression is achieved, but precise genome modification is required
Solution Approach 1:
The patent applies CRISPR/Cas9 editing at a specific, predetermined genomic locus upstream of the RTP1 gene, rather than performing random mutagenesis or using broad-spectrum methods. The guide RNA is designed to target a specific sequence at the RTP1 locus, ensuring that the constitutive promoter is integrated at the correct location to activate endogenous expression. This localized precision is achieved through the sequence-specific binding of the guide RNA-Cas9 complex to the target locus, enabling efficient and accurate gene activation without affecting other genomic regions.
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 allows for the stable and efficient expression of the RTP1 protein in non-olfactory cells, significantly improving odorant receptor function, enabling high-throughput screening and comprehensive characterization of odorant interactions, and facilitating the discovery of malodour counteractants and new fragrance compounds.
Implementation Method 1
CRISPR/Cas9 is a highly efficient genome editing tool used to generate precise genome modifications such as insertions and deletions. By incorporating a transcriptional promoter upstream of the gene, a cell line may then express an endogenous gene that is otherwise inactive.
Implementation Method 2
By incorporating a transcriptional promoter upstream of the gene, a cell line may then express an endogenous gene that is otherwise inactive.
Data Source
AI summary
Provided herein is a cell line with improved odorant receptor function comprising an activated endogenous RTP1 gene, which further expresses an RTP1 protein. Further provided herein is a method for specifically activating an endogenous RTP1 gene in a eukaryotic cell using a CRISPR/Cas9 derived technique. Also provided herein is a method for identifying compounds with desired effects such as perfume or aroma modulators in said cell line.