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

• Main Authors: Mert Yanik (Author), Surya Prakash Goud Ponnam (Author), Tobias Wimmer (Author), Lennart Trimborn (Author), Carina Müller (Author), Isabel Gambert (Author), Johanna Ginsberg (Author), Annabella Janise (Author), Janina Domicke (Author), Wolfgang Wende (Author), Birgit Lorenz (Author), Knut Stieger (Author)
• Format: Book
• Published: Elsevier, 2018-06-01T00:00:00Z.
• Subjects: article
• Online Access: Connect to this object online.
• Notes: 2162-2531; 10.1016/j.omtn.2018.03.010
• Keywords: genome editing; CRISPR-Cas; MMEJ; XLRP; Csy4; Therapeutics. Pharmacology; RM1-950
• Source: Molecular Therapy: Nucleic Acids, Vol 11, Iss C, Pp 407-415 (2018)

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.