Journal article

Publication year: 2018

Pages: 407-415

Journal: Molecular Therapy: Nucleic Acids

Volume number: 11

ISSN: 2162-2531

Open access status: Gold

DOI Link: https://doi.org/10.1016/j.omtn.2018.03.010

Publisher: Cell Press

Abstract: 
Common genome-editing strategies are either based on nonhomologous end joining (NHEJ) or, in the presence of a template DNA, based on homologous recombination with long (homology-directed repair [HDR]) or short (microhomologymediated end joining [MMEJ]) homologous sequences. In the current study, we aim to develop a model system to test the activity of MMEJ after CRISPR/Cas9-mediated cleavage in cell culture. Following successful proof of concept in an episomally based reporter system, we tested template plasmids containing a promoter-less luciferase gene flanked by microhomologous sequences (mhs) of different length (5, 10, 15, 20, 30, and 50 bp) that are complementary to the mouse retinitis pigmentosa GTPase regulator (RPGR)-ORF15, which is under the control of a CMV promoter stably integrated into a HEK293 cell line. Luciferase signal appearance represented successful recombination events and was highest when the mhs were 5 bp long, while longer mhs revealed lower luciferase signal. In addition, presence of Csy4 RNase was shown to increase luciferase signaling. The luciferase reporter system is a valuable tool to study the input of the different DNA repair mechanisms in the replacement of large DNA sequences by mhs.

Harvard Citation style: Yanik, M., Ponnam, S., Wimmer, T., Trimborn, L., Mueller, C., Gambert, I., et al. (2018) Development of a Reporter System to Explore MMEJ in the Context of Replacing Large Genomic Fragments, Molecular Therapy: Nucleic Acids, 11, pp. 407-415. https://doi.org/10.1016/j.omtn.2018.03.010

APA Citation style: Yanik, M., Ponnam, S., Wimmer, T., Trimborn, L., Mueller, C., Gambert, I., Ginsberg, J., Janise, A., Domicke, J., Wende, W., Lorenz, B., & Stieger, K. (2018). Development of a Reporter System to Explore MMEJ in the Context of Replacing Large Genomic Fragments. Molecular Therapy: Nucleic Acids. 11, 407-415. https://doi.org/10.1016/j.omtn.2018.03.010