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

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.

Understanding the diversity of genetic outcomes from CRISPR-Cas generated homology-directed repair

“…Multiple pathways of repair are active simultaneously on a cleaved DNA molecule. In the absence of donor DNA, some form of NHEJ and/or MMEJ 27 reconnects the linear plasmid template; in some cases, termini resection generates a deletion at the target site. In the presence of donor DNA, some form of homology-directed repair takes place potentially leading to precise or error-prone repair.…”

“…The cell-free system has been shown to produce deletions, insertions, and precise and error-prone repair through several recombinational repair pathways 22,23 . The insertion of small fragments is likely catalyzed by the process of MMEJ 27 while deletions in target DNA are likely the result of resection and NHEJ 13 .…”

Microhomologies are prevalent at Cas9-induced larger deletions

“…MMEJ has previously been implicated in targeted insertions (77–80) as well as deletions, but in previous studies was associated with smaller deletions than the LDs we observed (7,15,39,46,55–61). MMEJ has also been implicated in chromosomal translocations (7,81,82), which by their nature take place over large genomic sequence scales. A recent study found that MMEJ was most active with 5 bp microhomologies (81), which is in line with the overrepresented short microhomologies we observed at Cas9-induced LDs.…”

“…MMEJ has also been implicated in chromosomal translocations (7,81,82), which by their nature take place over large genomic sequence scales. A recent study found that MMEJ was most active with 5 bp microhomologies (81), which is in line with the overrepresented short microhomologies we observed at Cas9-induced LDs. Moreover, MMEJ repair is thought to favour GC base paring (46), which we also observed at Cas9-induced LDs.…”

Using Transcriptomic Analysis to Assess Double-Strand Break Repair Activity: Towards Precise in Vivo Genome Editing

“…To this end, in vivo genome editing represents a promising emerging treatment of such monogenic disorders, as it aims to correct the disease-causing mutation within the genome, resulting in the restoration of endogenous protein production [9]. To target a large number of disease-causing mutations within the same gene, replacing large DNA fragments comprising several exons might be advantageous over targeting single mutations (Figure 1c) [10].…”