CRISPR-Cas9-Mediated Bioluminescent Tagging of Endogenous Proteins by Fluorescent Protein-Assisted Cell Sorting

Area of Science:

• Oncology
• Molecular Biology
• Biochemistry

Background:

• Oncogenic fusion genes are crucial drivers in many cancers, presenting attractive therapeutic targets.
• Transcription factor fusions like PAX3-FOXO1 in alveolar fusion gene-positive rhabdomyosarcoma (FP-RMS) lack obvious drug-binding sites, hindering targeted inhibition.
• Existing therapies targeting kinases, such as imatinib mesylate for BCR-ABL1, are not directly applicable to transcription factor fusions.

Purpose of the Study:

• To identify novel small molecules that selectively target the PAX3-FOXO1 fusion protein.
• To develop a sensitive and quantitative method for screening compound libraries for PAX3-FOXO1 inhibitors and degraders.

Main Methods:

• CRISPR-Cas9-mediated knock-in was used to tag the endogenous PAX3-FOXO1 fusion protein with a pro-luminescent HiBiT tag in FP-RMS cell lines.
• A P2A self-cleaving peptide linked to an mCherry fluorescent protein was included for fluorescence-activated cell sorting (FACS)-based selection of single-cell clones.
• The HiBiT tag enables highly sensitive luminescence detection of PAX3-FOXO1 protein levels.

Main Results:

• Successful generation of FP-RMS cell lines with a carboxy-terminal HiBiT tag on the endogenous PAX3-FOXO1 fusion protein.
• The HiBiT tag provides a ~100-fold brighter luminescence signal compared to conventional luciferases.
• The developed system allows for quantitative, high-throughput screening of compound libraries.

Conclusions:

• HiBiT tagging of PAX3-FOXO1 provides a sensitive and robust platform for discovering novel therapeutic agents against FP-RMS.
• This approach overcomes the challenge of targeting transcription factor fusions by enabling the detection of protein levels.
• The methodology facilitates the identification of both PAX3-FOXO1 inhibitors and degraders for potential cancer treatment.