Regensburg 2022 – wissenschaftliches Programm
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BP: Fachverband Biologische Physik
BP 12: Poster 2
BP 12.59: Poster
Dienstag, 6. September 2022, 17:30–19:30, P4
Single Cell Prime Editing Kinetics — •Nathalie Schäffler1, Julian Geilenkeuser2, Dong-Jiunn Jeffery Truong2, Gil Westmeyer2, and Joachim Rädler1 — 1LMU München, Deutschland — 2Institute for Synthetic Biomedicine, Helmholtz-Zentrum München, Deutschland
CRISPR-Cas technology opens up new ways of approaching biological computing, taking advantage of the native language of biology and the inherent possibilities of DNA which could enable easier parallelism and higher storage capacities. However, to effectively leverage this technology a solid understanding of the kinetics and efficiency of gene editing is essential.
A key advancement in CRISPR-Cas is Prime Editing (PE), which enables precise "search-and-replace" of specific DNA sections without templates. However, PE requires delivery of both the PE specific Cas-9 protein and a guide RNA (pegRNA) into living cells. Two common strategies of non-viral in vivo delivery are via mRNA or pDNA constructs encoding both PE components. We compare these two methods and study their efficiency and timing using Live Imaging on Single Cell Arrays (LISCA).
Our experiments use a HEK293T cell line with stable expressing blue shifted mGreenLantern (mGL) as reporter system, taking advantage of the fact that only a short DNA sequence edit is needed to reverse the blue shift back to the green mGL. By recording the single cell kinetics and statistics of PE converting bs-mGL into mGL starting from the time point of transfection, we can assess editing times and efficiencies.