Miyaoka Yuichiro, Berman Jennifer R, Cooper Samantha B, Mayerl Steven J, Chan Amanda H, Zhang Bin, Karlin-Neumann George A, Conklin Bruce R
Gladstone Institute of Cardiovascular Disease, San Francisco, California, 94158, USA.
Digital Biology Center, Bio-Rad Laboratories, Pleasanton, California, 94588, USA.
Sci Rep. 2016 Mar 31;6:23549. doi: 10.1038/srep23549.
Precise genome-editing relies on the repair of sequence-specific nuclease-induced DNA nicking or double-strand breaks (DSBs) by homology-directed repair (HDR). However, nonhomologous end-joining (NHEJ), an error-prone repair, acts concurrently, reducing the rate of high-fidelity edits. The identification of genome-editing conditions that favor HDR over NHEJ has been hindered by the lack of a simple method to measure HDR and NHEJ directly and simultaneously at endogenous loci. To overcome this challenge, we developed a novel, rapid, digital PCR-based assay that can simultaneously detect one HDR or NHEJ event out of 1,000 copies of the genome. Using this assay, we systematically monitored genome-editing outcomes of CRISPR-associated protein 9 (Cas9), Cas9 nickases, catalytically dead Cas9 fused to FokI, and transcription activator-like effector nuclease at three disease-associated endogenous gene loci in HEK293T cells, HeLa cells, and human induced pluripotent stem cells. Although it is widely thought that NHEJ generally occurs more often than HDR, we found that more HDR than NHEJ was induced under multiple conditions. Surprisingly, the HDR/NHEJ ratios were highly dependent on gene locus, nuclease platform, and cell type. The new assay system, and our findings based on it, will enable mechanistic studies of genome-editing and help improve genome-editing technology.
精确的基因组编辑依赖于通过同源定向修复（HDR）修复序列特异性核酸酶诱导的DNA切口或双链断裂（DSB）。然而，易错的非同源末端连接（NHEJ）同时起作用，降低了高保真编辑的效率。由于缺乏一种在内源基因座直接同时测量HDR和NHEJ的简单方法，有利于HDR而非NHEJ的基因组编辑条件的鉴定受到了阻碍。为了克服这一挑战，我们开发了一种基于数字PCR的新型快速检测方法，该方法可以在1000份基因组拷贝中同时检测到一个HDR或NHEJ事件。使用该检测方法，我们系统地监测了CRISPR相关蛋白9（Cas9）、Cas9切口酶、与FokI融合的催化失活Cas9以及转录激活样效应核酸酶在HEK293T细胞、HeLa细胞和人诱导多能干细胞中三个疾病相关内源基因座的基因组编辑结果。尽管人们普遍认为NHEJ通常比HDR更常发生，但我们发现在多种条件下诱导的HDR多于NHEJ。令人惊讶的是，HDR/NHEJ比率高度依赖于基因座、核酸酶平台和细胞类型。新的检测系统以及我们基于此的发现将有助于进行基因组编辑的机制研究，并有助于改进基因组编辑技术。
