A Rapid and Precise Mutation-Activated Fluorescence Reporter for Analyzing Acute Mutagenesis Frequency

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Current mutagenesis reporters often rely on cell survival, leading to lengthy experimental times (weeks) and limiting the assessment of acute or time-dependent mutagenic events.
• Existing methods for quantifying genome stability can be restricted by cell type and do not allow for real-time mutation monitoring.

Purpose of the Study:

• To develop a novel, rapid, and versatile biosensor for quantifying mutagenesis and genome stability.
• To overcome the limitations of current methods, including long assay times and cell-type specificity.

Main Methods:

• Engineered a mutation biosensor (CherryOFF-GFP) by modifying mCherryFP to be fluorescence-dependent on a specific codon (Trp98).
• The biosensor's red fluorescence is activated by a specific A/T-G/C nucleotide transition, with GFP serving as an internal control.
• Compared the CherryOFF-GFP reporter's performance against the established hypoxanthine phosphoribosyl transferase assay.

Main Results:

• The CherryOFF-GFP biosensor detects specific A/T-G/C nucleotide transitions, activating red fluorescence.
• The reporter demonstrates comparable or superior sensitivity to the hypoxanthine phosphoribosyl transferase assay in detecting mutation frequency changes.
• CherryOFF-GFP provides readouts within one day, independent of cell-death events, and is adaptable to various cell types.

Conclusions:

• CherryOFF-GFP offers a significant advancement in mutagenesis detection, enabling rapid, cell-type-independent quantification of genome instability.
• This novel reporter facilitates the study of acute and time-dependent mutagenic events, overcoming key limitations of existing assays.