Research Centre for Medical Genetics, Moskvorechie 1, 115522 Moscow, Russia.
Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Pirogov Russian National Research Medical University, Ostrovityanova 1, 117997 Moscow, Russia.
Int J Mol Sci. 2023 Apr 4;24(7):6704. doi: 10.3390/ijms24076704.
This study aimed to enhance homology-directed repair (HDR) efficiency in CRISPR/Cas-mediated genome editing by targeting three key factors regulating the balance between HDR and non-homologous end joining (NHEJ): MAD2L2, SCAI, and Ligase IV. In order to achieve this, a cellular model using mutated eGFP was designed to monitor HDR events. Results showed that MAD2L2 knockdown and SCR7 treatment significantly improved HDR efficiency during Cas9-mediated HDR repair of the mutated eGFP gene in the HEK293T cell line. Fusion protein Cas9-SCAI did not improve HDR. This study is the first to demonstrate that MAD2L2 knockdown during CRISPR-mediated gene editing in HEK293T cells can increase precise correction by up to 10.2 times. The study also confirmed a moderate but consistent effect of SCR7, an inhibitor of Ligase IV, which increased HDR by 1.7 times. These findings provide valuable insights into improving HDR-based genome editing efficiency.
本研究旨在通过靶向调节同源重组 (HDR) 和非同源末端连接 (NHEJ) 之间平衡的三个关键因素：MAD2L2、SCAI 和 Ligase IV，来提高 CRISPR/Cas 介导的基因组编辑中的 HDR 效率。为了实现这一目标，设计了一个使用突变型 eGFP 的细胞模型来监测 HDR 事件。结果表明，在 Cas9 介导的突变型 eGFP 基因的 HEK293T 细胞系 HDR 修复过程中，MAD2L2 敲低和 SCR7 处理显著提高了 HDR 效率。融合蛋白 Cas9-SCAI 并没有提高 HDR。这项研究首次证明，在 HEK293T 细胞中进行 CRISPR 介导的基因编辑时，MAD2L2 的敲低可以将精确修复提高多达 10.2 倍。该研究还证实了 Ligase IV 抑制剂 SCR7 的适度但一致的作用，它将 HDR 提高了 1.7 倍。这些发现为提高基于 HDR 的基因组编辑效率提供了有价值的见解。
