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 non-homologous end-joining (NHEJ). However, the low efficiency of CRISPR-del results in laborious experiments and false negative results. Using an endogenous reporter system, we demonstrate that temporary inhibition of DNA-dependent protein kinase (DNA-PK) - an early step in NHEJ - yields up to 17-fold increase 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. Importantly, the method is compatible with pooled functional screens employing lentivirally-delivered guide RNAs. Thus, delaying the kinetics of NHEJ relative to DSB formation is a simple and effective means of enhancing CRISPR-deletion.

中文翻译：

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通过DNA-PK的药理学延迟增强CRISPR缺失

CRISPR-Cas9缺失（CRISPR-del）是从哺乳动物细胞中消除DNA的主要方法，并支持各种基因组编辑应用。由一对双链断裂（DSB）定义的靶DNA在非同源末端连接（NHEJ）期间被去除。然而，CRISPR-del的低效率导致费力的实验和假阴性结果。使用内源性报告系统，我们证明对DNA依赖性蛋白激酶（DNA-PK）的暂时抑制-NHEJ的早期步骤-产生高达17倍的DNA缺失增加。在多种细胞系，基因传递方法，商业抑制剂和指导RNA（包括那些原本可以忽略不计的活性）中观察到了这一点。重要的是，该方法与采用慢病毒递送的指导RNA的合并功能筛选兼容。从而，