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.

常见的基因组编辑策略要么基于非同源末端连接 (NHEJ)，要么在存在模板 DNA 的情况下，基于长（同源定向修复 [HDR]）或短（微同源介导的末端连接）的同源重组[MMEJ]) 同源序列。在目前的研究中，我们的目标是开发一个模型系统来测试细胞培养中 CRISPR/Cas9 介导的裂解后 MMEJ 的活性。在基于附加型的报告系统中成功证明概念后，我们测试了包含无启动子荧光素酶基因的模板质粒，其两侧是不同长度（5、10、15、20、30 和 50 bp）的微同源序列 (mhs)，它们是补充小鼠视网膜色素变性GTPase 调节器 (RPGR)-ORF15，在 CMV 启动子的控制下稳定整合到 HEK293 细胞系中。荧光素酶信号出现代表成功的重组事件，当 mhs 为 5 bp 时最高，而较长的 mhs 显示较低的荧光素酶信号。此外，Csy4 RNase 的存在显示出增加荧光素酶信号。荧光素酶报告系统是研究不同 DNA 修复机制在 mhs 替换大 DNA 序列中的输入的宝贵工具。