SKM 2023 – wissenschaftliches Programm

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BP: Fachverband Biologische Physik

BP 11: Poster Session I

BP 11.71: Poster

Dienstag, 28. März 2023, 12:30–15:30, P1

The Influence of pegRNA Variations on Prime Editing Kinetics — •Nathalie Schäffler¹, Julian Geilenkeuser², Dong-Jiunn Jeffery Truong², Gil Westmeyer², and Joachim Rädler¹ — ¹LMU München, Deutschland — ²ISBM, Helmholtz-Zentrum München, Deutschland

A key development in recent CRISPR technology is the "Search and Replace" system, also called Prime Editing (PE). Right now, the optimization of this is an active research field, which could lead to interesting new possibilities like approaches for biocomputing. However, current studies focus primarily on endpoint measurements with FACS.

In our research we compare different pegRNA variations and their editing efficiency via collective and single cell timelapse measurements. We transfect a HEK293T cell line, which stably expresses a blue shifted mGreenLantern (bs-mGL), with the two PE components: pegRNA and Cas9-complex. With this, the cells gain the ability to edit their bsmGL DNA back to the original green mGL sequence. We track the fluorescence-time courses of this signal with Live-cell Imaging of Single Cell Arrays (LISCA) and use kinetic rate equations to better understand the defining processes in the timing of those edits.

This allows us to have a closer look into the kinetics of PE.