A transient reporter for editing enrichment (TREE) in human cells

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Current methods for identifying base-edited cells are inefficient and require sequencing.
• Deaminase base editing technologies offer precise genetic modifications but lack efficient cellular reporting systems.

Purpose of the Study:

• To develop a novel, efficient assay for direct reporting of base editing activity within cells.
• To optimize base editing parameters and purification strategies for enriched cell populations.
• To enable efficient base editing in human pluripotent stem cells (hPSCs).

Main Methods:

• Utilized a blue fluorescent protein (BFP) to green fluorescent protein (GFP) conversion assay reporting C-to-T substitutions.
• Developed the transient reporter for editing enrichment (TREE) assay using flow cytometry for cell purification.
• Optimized base editor vector design and transfection parameters in hPSCs.

Main Results:

• The TREE assay achieved purification efficiencies approaching 80% for base-edited cell populations.
• Demonstrated significantly improved editing efficiency compared to conventional reporters of transfection (RoT).
• Successfully optimized base editor vector design and achieved highly efficient editing in hPSCs, a previously resistant cell type.

Conclusions:

• The TREE assay provides a robust and efficient method for identifying and enriching base-edited cells.
• This technology facilitates base editing applications in synthetic biology, disease modeling, and regenerative medicine.
• The developed methods enable efficient genome editing in challenging cell types like hPSCs.