CRISPR-Edited Cell Lines for Odorant Receptor Screening

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Solution Overview

Problem

Current methods for developing stable cell lines that express the endogenous RTP1 gene are inefficient and cumbersome, and there is a need for a reliable way to functionally express odorant receptors on non-olfactory cell surfaces for high-throughput screening of odorant and aroma compounds.

Innovation Solution

The use of CRISPR/Cas9 genome editing to introduce a promoter upstream of the RTP1 gene locus in mammalian cell lines, ensuring consistent expression of the RTP1 protein, particularly the short version (RTP1S), which enhances odorant receptor function and allows for comprehensive characterization of odorant-receptor interactions.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If traditional molecular biology approaches are used to insert DNA into cultured cells to stably express the endogenous RTP1 gene, then the cell line can be modified to express RTP1, but the process is inefficient and cumbersome

Engineering Contradiction:

Improvestable expression of RTP1 geneVSAvoidefficiency of cell line development

Core Design Contradiction:

ReliabilityVSProductivity

Solution Approach 1:

The patent replaces traditional mechanical DNA insertion methods (microinjection, electroporation, viral transduction) with a chemical/genetic approach using CRISPR-Cas9 mediated homologous recombination. This substitution eliminates the need for complex physical manipulation of DNA into cells, significantly simplifying the process while achieving stable RTP1 expression

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The patent introduces a donor DNA molecule as an intermediary that contains the promoter and RTP1 gene sequence. This donor DNA serves as a template that undergoes homologous recombination with the endogenous RTP1 locus, enabling stable integration and expression without direct DNA insertion methods

Inventive Principle:

Principle #24Intermediary (Mediator)

2Productivity

If odorant receptors are expressed on non-olfactory cell surfaces for high-throughput screening, then comprehensive characterization of OR activity is enabled, but reliable decoding of OR codes requires optimized cell surface expression

Engineering Contradiction:

Improvehigh-throughput screening capabilityVSAvoidaccuracy of OR code decoding

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent modifies the expression parameters of the RTP1 gene by introducing a constitutive promoter that drives high-level, stable expression. This parameter change ensures sufficient protein is produced at the cell surface to reliably support odorant receptor function and decoding accuracy during high-throughput screening

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent performs preliminary action by pre-establishing the cell line with the constitutively active RTP1 gene through CRISPR-Cas9 editing before beginning odorant receptor expression and screening experiments. This preliminary modification ensures that the cell line is ready for reliable, high-throughput OR characterization without requiring subsequent complex optimizations

Inventive Principle:

Principle #10Preliminary action

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 the creation of non-olfactory cell lines with improved odorant receptor function, facilitating the discovery of malodour counteractants, new flavor, and fragrance compounds by enabling precise and efficient expression of the RTP1 gene, thereby enhancing the decoding of odorant-receptor interactions.

Implementation Method 1

CRISPR/Cas9 is a highly efficient genome editing tool used to generate precise genome modifications such as insertions and deletions

Methodology Applied

Scientific EffectCRISPR/Cas9 genome editing:

Implementation Method 2

the donor DNA comprises a promoter wherein the promoter drives the expression of the RTP1 gene

Methodology Applied

Scientific EffectTranscription:

Implementation Method 3

This protein has been shown to act, in part, by interacting with odorant receptors in the endoplasmic reticulum

Methodology Applied

Scientific EffectTranslation:

Implementation Method 4

These proteins function by assisting in the trafficking of the odorant receptors from the endoplasmic reticulum to the Golgi apparatus and plasma membrane of the cell

Methodology Applied

Scientific EffectProtein trafficking:

Implementation Method 5

Odors are initially encoded in the peripheral olfactory system (i.e. the nose) through interactions between volatile flavor and fragrance compounds and odorant receptor (OR) proteins that reside on the membranes of olfactory receptor neurons

Methodology Applied

Scientific EffectReceptor-ligand interaction:

Data Source

PatentEP3726218B1Cell lines for screening odorant and aroma receptors

Publication Date: 2023.08.09 FIRMENICH SA

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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.