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Cellular thermal shift analysis for interrogation of CRISPR-assisted proteomic changes
Nam-Gu Her1,2, Ivan Babic3, Venkata M Yenugonda3
1Korea Institute of Radiological & Medical Sciences, Nowon, Seoul 01812, Korea.
Abstract:
CRISPR-Cas9 has proven to be a versatile tool for the discovery of essential genetic elements involved in various disease states. CRISPR-assisted dense mutagenesis focused on therapeutically challenging protein complexes allows us to systematically perturb protein-coding sequences in situ and correlate them with functional readouts. Such perturbations can mimic targeting by therapeutics and serve as a foundation for the discovery of highly specific modulators. However, translation of such genomics data has been challenging due to the missing link for proteomics under the physiological state of the cell. We present a method based on cellular thermal shift assays to easily interrogate proteomic shifts generated by CRISPR-assisted dense mutagenesis, as well as a case focused on NuRD epigenetic complex.
Insights
CRISPR-Cas9 gene editing enables dense mutagenesis to identify disease-related genes. A new method links these genetic changes to protein alterations, aiding therapeutic modulator discovery for complexes like NuRD.
Area of Science:
- Genomics and Proteomics
- Molecular Biology
- Epigenetics
Background:
- CRISPR-Cas9 is a powerful tool for genetic element discovery in diseases.
- Perturbing protein-coding sequences in situ aids in identifying therapeutic targets.
- Linking genomics data to cellular proteomics is crucial but challenging.
Purpose of the Study:
- To develop a method for interrogating proteomic shifts from CRISPR-assisted dense mutagenesis.
- To establish a link between genetic perturbations and proteomic changes in cells.
- To apply this method to study the NuRD epigenetic complex.
Main Methods:
- CRISPR-assisted dense mutagenesis to perturb protein-coding sequences.
- Cellular thermal shift assays (CETSA) to measure proteomic changes.
- Application of the method to the NuRD epigenetic complex.
Main Results:
- Successfully integrated CRISPR-based mutagenesis with proteomic analysis.
- Demonstrated the ability to interrogate proteomic shifts in response to genetic perturbations.
- Provided a case study on the NuRD epigenetic complex.
Conclusions:
- The developed method bridges the gap between genomics and proteomics for disease-related gene discovery.
- This approach facilitates the identification of modulators for challenging therapeutic targets.
- The study highlights the utility of combining CRISPR mutagenesis with CETSA for biological insights.
