Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, 3010 Bern, Switzerland.
Department for BioMedical Research, University of Bern, 3008 Bern, Switzerland.
Genome Res. 2021 Mar;31(3):461-471. doi: 10.1101/gr.265736.120. Epub 2021 Feb 11.
CRISPR-Cas9 deletion (CRISPR-del) is the leading approach for eliminating DNA from mammalian cells and underpins a variety of genome-editing applications. Target DNA, defined by a pair of double-strand breaks (DSBs), is removed during nonhomologous end-joining (NHEJ). However, the low efficiency of CRISPR-del results in laborious experiments and false-negative results. By using an endogenous reporter system, we show that repression of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-an early step in NHEJ-yields substantial increases in DNA deletion. This is observed across diverse cell lines, gene delivery methods, commercial inhibitors, and guide RNAs, including those that otherwise display negligible activity. We further show that DNA-PKcs inhibition can be used to boost the sensitivity of pooled functional screens and detect true-positive hits that would otherwise be overlooked. Thus, delaying the kinetics of NHEJ relative to DSB formation is a simple and effective means of enhancing CRISPR-deletion.
CRISPR-Cas9 缺失（CRISPR-del）是从哺乳动物细胞中去除 DNA 的主要方法，为各种基因组编辑应用提供了支持。靶 DNA 由双链断裂（DSB）定义，在非同源末端连接（NHEJ）过程中被去除。然而，CRISPR-del 的低效率导致实验繁琐且结果可能为假阴性。通过使用内源性报告系统，我们表明 DNA 依赖性蛋白激酶催化亚基（DNA-PKcs）的抑制——NHEJ 的早期步骤——可显著增加 DNA 缺失。这在多种细胞系、基因传递方法、商业抑制剂和向导 RNA 中均有观察到，包括那些原本显示出微不足道活性的抑制剂和向导 RNA。我们进一步表明，DNA-PKcs 抑制可用于提高池功能筛选的灵敏度，并检测到否则可能被忽略的真正阳性命中。因此，相对于 DSB 形成延迟 NHEJ 的动力学是增强 CRISPR-del 的一种简单有效的方法。
