Targeted Protein Degradation Phenotypic Studies Using HaloTag CRISPR/Cas9 Endogenous Tagging Coupled with HaloPROTAC3

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Protein loss studies are crucial for understanding protein function and identifying disease targets.
• Current methods like mutagenesis and CRISPR knockout are often irreversible and can cause cell death.
• A reversible and conditional approach is needed for studying essential proteins and therapeutic targets.

Purpose of the Study:

• To develop a reversible and conditional live-cell knockout strategy for studying protein function.
• To enable conditional degradation of endogenous proteins using a modular protein-tagging system.
• To provide a sensitive method for detecting protein degradation.

Main Methods:

• Developed a modular protein-tagging approach using HaloTag and HaloPROTAC3 for conditional protein degradation.
• Utilized CRISPR/Cas9 for genomic insertion of HaloTag at protein N or C termini.
• Employed Fluorescence-Activated Cell Sorting (FACS) for enrichment of edited cells.
• Integrated the HiBiT peptide tag for sensitive luminescence detection of HaloTag fusion levels.
• Compared HaloPROTAC3 degradation efficiency with dTAG-13.

Main Results:

• Successfully demonstrated reversible and conditional degradation of endogenous HaloTag fusion proteins in various subcellular locations.
• The HiBiT tag enabled sensitive, antibody-free detection of protein degradation.
• HaloPROTAC3 exhibited a faster degradation rate and comparable extent to dTAG-13.
• Provided protocols for HaloTag insertion and HaloPROTAC3-mediated degradation.

Conclusions:

• The HaloPROTAC3 system offers a versatile and efficient method for conditional protein knockout in live cells.
• This approach overcomes limitations of irreversible gene knockout methods, facilitating the study of essential genes.
• The developed strategy has significant implications for functional genomics and the discovery of therapeutic targets.