The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-associated proteins) technology enables rapid and precise genome editing at any desired genomic position in almost all cells and organisms. In this study, we analyzed the impact of different repair templates on the frequency of homology-directed repair (HDR) and non-homologous end joining (NHEJ). We used a stable HEK293 cell line expressing the traffic light reporter (TLR-3) system to quantify HDR and NHEJ events following transfection with Cas9, eight different guide RNAs, and a 1,000 bp donor template generated either as circular plasmid, as linearized plasmid with long 3' or 5' backbone overhang, or as PCR product. The sequence to be corrected was either centrally located (RS55), with a shorter 5' homologous region (RS37), or with a shorter 3' homologous region (RS73). Guide RNAs targeting the transcriptionally active strand (T5, T7) showed significantly higher NHEJ frequencies compared with guide RNAs targeting the transcriptionally inactive strand. HDR activity was highest when using the linearized plasmid with the short 5' backbone overhang and the RS37 design. The results demonstrate the importance of the design of the guide RNA and template DNA on the frequency of DNA repair events and, ultimately, on the outcome of treatment approaches using HDR.

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.