Radiation Biology and DNA Repair, Technical University of Darmstadt, 64287 Darmstadt, Germany.
Plant Membrane Biophysics, Technical University of Darmstadt, 64287 Darmstadt, Germany.
Cells. 2020 Apr 12;9(4):947. doi: 10.3390/cells9040947.
Genome editing (GE) represents a powerful approach to fight inherited blinding diseases in which the underlying mutations cause the degeneration of the light sensing photoreceptor cells of the retina. Successful GE requires the efficient repair of DNA double-stranded breaks (DSBs) generated during the treatment. Rod photoreceptors of adult mice have a highly specialized chromatin organization, do not efficiently express a variety of DSB response genes and repair DSBs very inefficiently. The DSB repair efficiency in rods of other species including humans is unknown. Here, we used ionizing radiation to analyze the DSB response in rods of various nocturnal and diurnal species, including genetically modified mice, pigs, and humans. We show that the inefficient repair of DSBs in adult mouse rods does not result from their specialized chromatin organization. Instead, the DSB repair efficiency in rods correlates with the level of Kruppel-associated protein-1 (KAP1) expression and its ataxia-telangiectasia mutated (ATM)-dependent phosphorylation. Strikingly, we detected robust KAP1 expression and phosphorylation only in human rods but not in rods of other diurnal species including pigs. Hence, our study provides important information about the uniqueness of the DSB response in human rods which needs to be considered when choosing model systems for the development of GE strategies.
基因组编辑(GE)代表了一种对抗遗传性致盲疾病的强大方法,这些疾病的潜在突变导致视网膜感光细胞的退化。成功的 GE 需要在治疗过程中产生的 DNA 双链断裂(DSB)的有效修复。成年小鼠的杆状光感受器具有高度特化的染色质组织,不能有效地表达多种 DSB 反应基因,并且修复 DSB 的效率非常低。其他物种(包括人类)的杆状细胞中的 DSB 修复效率尚不清楚。在这里,我们使用电离辐射来分析包括遗传修饰小鼠、猪和人类在内的各种夜间和日间物种的杆状细胞中的 DSB 反应。我们表明,成年小鼠杆状细胞中 DSB 修复效率低下并不是由于其特化的染色质组织造成的。相反,杆状细胞中 DSB 修复效率与 Kruppel 相关蛋白-1(KAP1)表达水平及其共济失调毛细血管扩张突变(ATM)依赖性磷酸化相关。引人注目的是,我们仅在人类杆状细胞中检测到了强大的 KAP1 表达和磷酸化,而在其他日间物种(包括猪)的杆状细胞中则没有。因此,我们的研究提供了有关人类杆状细胞 DSB 反应独特性的重要信息,在选择用于开发 GE 策略的模型系统时需要考虑这些信息。