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Development of a Reporter System to Explore MMEJ in the Context of Replacing Large Genomic Fragments

Yanik, Mert ; Ponnam, Surya Prakash Goud ; Wimmer, Tobias ; Trimborn, Lennart ; M├╝ller, Carina ; Gambert, Isabel ; Ginsberg, Johanna ; Janise, Annabella ; Domicke, Janina ; Wende, Wolfgang ; Lorenz, Birgit ; Stieger, Knut

Originalveröffentlichung: (2018) Molecular Therapy - Nucleic Acids 11:407-415 doi:
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URN: urn:nbn:de:hebis:26-opus-139892

Sammlung: Open Access - Publikationsfonds
Universität Justus-Liebig-Universit├Ąt Gie├čen
Institut: Department of Ophthalmology
Fachgebiet: Medizin
DDC-Sachgruppe: Medizin
Dokumentart: Aufsatz
Sprache: Englisch
Erstellungsjahr: 2018
Publikationsdatum: 31.01.2019
Kurzfassung auf Englisch: Common genome-editing strategies are either based on non-homologous end joining (NHEJ) or, in the presence of a template DNA, based on homologous recombination with long (homology-directed repair [HDR]) or short (microhomology-mediated 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.
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