Development of a Reporter System to Explore MMEJ in the Context of Replacing Large Genomic Fragments
dc.contributor.author | Yanik, Mert | |
dc.contributor.author | Ponnam, Surya Prakash Goud | |
dc.contributor.author | Wimmer, Tobias | |
dc.contributor.author | Trimborn, Lennart | |
dc.contributor.author | Müller, Carina | |
dc.contributor.author | Gambert, Isabel | |
dc.contributor.author | Ginsberg, Johanna | |
dc.contributor.author | Janise, Annabella | |
dc.contributor.author | Domicke, Janina | |
dc.contributor.author | Wende, Wolfgang | |
dc.contributor.author | Lorenz, Birgit | |
dc.contributor.author | Stieger, Knut | |
dc.date.accessioned | 2022-11-18T09:52:58Z | |
dc.date.available | 2019-01-31T12:36:40Z | |
dc.date.available | 2022-11-18T09:52:58Z | |
dc.date.issued | 2018 | |
dc.description.abstract | 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. | en |
dc.identifier.uri | http://nbn-resolving.de/urn:nbn:de:hebis:26-opus-139892 | |
dc.identifier.uri | https://jlupub.ub.uni-giessen.de//handle/jlupub/9395 | |
dc.identifier.uri | http://dx.doi.org/10.22029/jlupub-8783 | |
dc.language.iso | en | de_DE |
dc.rights | Namensnennung, Nicht kommerziell, keine Bearbeitung 4.0 International | * |
dc.rights.uri | https://creativecommons.org/licenses/by-nc-nd/4.0/ | * |
dc.subject.ddc | ddc:610 | de_DE |
dc.title | Development of a Reporter System to Explore MMEJ in the Context of Replacing Large Genomic Fragments | en |
dc.type | article | de_DE |
local.affiliation | FB 11 - Medizin | de_DE |
local.opus.fachgebiet | Medizin | de_DE |
local.opus.id | 13989 | |
local.opus.institute | Department of Ophthalmology | de_DE |
local.source.freetext | Molecular Therapy - Nucleic Acids 11:407-415 | de_DE |
local.source.uri | https://doi.org/https://doi.org/10.1016/j.omtn.2018.03.010 |
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