GO: a functional reporter system to identify and enrich base editing activity

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Base editing (BE) enables precise single nucleotide variant (SNV) engineering in various model systems.
• Identifying and isolating base-edited cells remains a significant challenge in genome modification research.
• Advancements in base editing enzymes offer a broader toolkit for genetic manipulation.

Purpose of the Study:

• To develop a sensitive and specific reporter system for in situ detection of base editing events.
• To evaluate the performance of novel base editing enzymes using the developed reporter system.
• To demonstrate the versatility of the reporter system for various downstream applications in biomedical research.

Main Methods:

• Development of a 'Gene On' (GO) reporter system utilizing an activatable Green Fluorescent Protein (GFP).
• Application of the GO system to assess kinetics, efficiency, and PAM specificity of diverse base editing variants.
• Adaptation of the GO system for inducible expression of genetic markers, antibiotic resistance, and enzymes like Cre recombinase.

Main Results:

• The GO reporter system accurately indicates cytosine and adenine base editing in situ with high sensitivity and specificity.
• The system facilitated the characterization of new base editing enzyme variants.
• GO demonstrated flexibility in linking base editing events to cellular functions via various reporter outputs.

Conclusions:

• The 'Gene On' reporter system significantly enhances the practicality and feasibility of CRISPR base editing.
• This tool aids in the identification and isolation of edited cells, streamlining biomedical research.
• GO enables functional linkage of base editing at endogenous loci to cellular activities in diverse cell models.