School of Biosciences, University of Kent, Canterbury CT2 7NJ, UK.
Department of Oncology and Metabolism, University of Sheffield, Sheffield S10 2RX, UK.
Biomolecules. 2021 Feb 15;11(2):288. doi: 10.3390/biom11020288.
Mammalian cells are constantly subjected to a variety of DNA damaging events that lead to the activation of DNA repair pathways. Understanding the molecular mechanisms of the DNA damage response allows the development of therapeutics which target elements of these pathways. Double-strand breaks (DSB) are particularly deleterious to cell viability and genome stability. Typically, DSB repair is studied using DNA damaging agents such as ionising irradiation or genotoxic drugs. These induce random lesions at non-predictive genome sites, where damage dosage is difficult to control. Such interventions are unsuitable for studying how different DNA damage recognition and repair pathways are invoked at specific DSB sites in relation to the local chromatin state. The RNA-guided Cas9 (CRISPR-associated protein 9) endonuclease enzyme is a powerful tool to mediate targeted genome alterations. Cas9-based genomic intervention is attained through DSB formation in the genomic area of interest. Here, we have harnessed the power to induce DSBs at defined quantities and locations across the human genome, using custom-designed promiscuous guide RNAs, based on in silico predictions. This was achieved using electroporation of recombinant Cas9-guide complex, which provides a generic, low-cost and rapid methodology for inducing controlled DNA damage in cell culture models.
哺乳动物细胞经常受到各种导致 DNA 修复途径激活的 DNA 损伤事件的影响。了解 DNA 损伤反应的分子机制可以开发针对这些途径的靶向治疗方法。双链断裂 (DSB) 对细胞活力和基因组稳定性特别有害。通常，使用电离辐射或遗传毒性药物等 DNA 损伤剂来研究 DSB 修复。这些药物在不可预测的基因组位点诱导随机损伤，其中损伤剂量难以控制。这种干预措施不适合研究在特定 DSB 位点与局部染色质状态相关的不同 DNA 损伤识别和修复途径如何被调用。RNA 引导的 Cas9（CRISPR 相关蛋白 9）内切酶是一种介导靶向基因组改变的强大工具。基于 Cas9 的基因组干预是通过在感兴趣的基因组区域形成 DSB 来实现的。在这里，我们利用基于计算机预测的定制通用向导 RNA，在整个人类基因组中以定义的数量和位置诱导 DSB，从而实现了这一目标。这是通过重组 Cas9 指导复合物的电穿孔来实现的，这为细胞培养模型中的诱导可控 DNA 损伤提供了一种通用、低成本和快速的方法。
