Averina O A, Kuznetsova S A, Permyakov O A, Sergiev P V
Institute of Functional Genomics, Moscow State University, Moscow, 119991 Russia.
Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, 119991 Russia.
Mol Biol (Mosk). 2024 Jul-Aug;58(4):525-548.
The CRISPR/Cas technology of targeted genome editing made it possible to carry out genetic engineering manipulations with eukaryotic genomes with a high efficiency. Targeted induction of site-specific DNA breaks is one of the key stages of the technology. The cell repairs the breaks via one of the two pathways, nonhomologous end joining (NHEJ) and homology-driven repair (HDR). The choice of the DNA repair pathway is determined by the architecture of the DNA break region formed as a result of terminal resection and depends on the cell cycle phase. NHEJ is the main pathway of double-strand break (DSB) repair in mammalian cells and involves a nonspecific ligation reaction. The reaction accuracy depends on the structure of break ends, and various insertions or deletions may arise as a result in the target genome region. Integration of a necessary sequence into the genome occurs via HDR, which requires a template with homology regions flanking a DSB. Introducing a genetic construct into a particular genomic locus is an important task, but is currently intricate and laborious to perform. However, the choice of the repair pathway can be of principal importance for basic research of gene functions and construction of animal models of human diseases to develop therapies. The review summarizes and systematizes the available information on strategies designed to increase the HDR efficiency. The strategies that most efficiently shift the balance towards HDR include use of NHEJ inhibitors, regulation of the key factors of homologous recombination, control of the cell cycle and chromatin status, and construction of HDR templates.
靶向基因组编辑的CRISPR/Cas技术使高效地对真核生物基因组进行基因工程操作成为可能。靶向诱导位点特异性DNA断裂是该技术的关键步骤之一。细胞通过非同源末端连接（NHEJ）和同源性驱动修复（HDR）这两种途径之一来修复断裂。DNA修复途径的选择取决于末端切除后形成的DNA断裂区域的结构，并取决于细胞周期阶段。NHEJ是哺乳动物细胞中双链断裂（DSB）修复的主要途径，涉及非特异性连接反应。反应准确性取决于断裂末端的结构，结果可能会在目标基因组区域出现各种插入或缺失。通过HDR将所需序列整合到基因组中，这需要一个在DSB两侧具有同源区域的模板。将基因构建体引入特定基因组位点是一项重要任务，但目前执行起来复杂且费力。然而，修复途径的选择对于基因功能的基础研究以及构建人类疾病动物模型以开发治疗方法可能至关重要。这篇综述总结并系统化了有关旨在提高HDR效率的策略的现有信息。最有效地将平衡转向HDR的策略包括使用NHEJ抑制剂、调节同源重组的关键因子、控制细胞周期和染色质状态以及构建HDR模板。
