Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA.
Graduate Program in Biophysics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Nat Commun. 2017 May 12;8:15058. doi: 10.1038/ncomms15058.
Precisely measuring the location and frequency of DNA double-strand breaks (DSBs) along the genome is instrumental to understanding genomic fragility, but current methods are limited in versatility, sensitivity or practicality. Here we present Breaks Labeling In Situ and Sequencing (BLISS), featuring the following: (1) direct labelling of DSBs in fixed cells or tissue sections on a solid surface; (2) low-input requirement by linear amplification of tagged DSBs by in vitro transcription; (3) quantification of DSBs through unique molecular identifiers; and (4) easy scalability and multiplexing. We apply BLISS to profile endogenous and exogenous DSBs in low-input samples of cancer cells, embryonic stem cells and liver tissue. We demonstrate the sensitivity of BLISS by assessing the genome-wide off-target activity of two CRISPR-associated RNA-guided endonucleases, Cas9 and Cpf1, observing that Cpf1 has higher specificity than Cas9. Our results establish BLISS as a versatile, sensitive and efficient method for genome-wide DSB mapping in many applications.
精确测量基因组中 DNA 双链断裂 (DSB) 的位置和频率对于理解基因组的脆弱性至关重要，但目前的方法在通用性、灵敏度或实用性方面存在局限性。在这里，我们提出了 Breaks Labeling In Situ and Sequencing (BLISS)，具有以下特点：(1) 在固体表面上的固定细胞或组织切片中直接标记 DSB；(2) 通过体外转录对标记的 DSB 进行线性扩增，从而降低输入要求；(3) 通过独特的分子标识符进行 DSB 的定量；以及 (4) 易于扩展和多重化。我们应用 BLISS 来分析低输入量的癌细胞、胚胎干细胞和肝组织中内源性和外源性 DSB。我们通过评估两种 CRISPR 相关的 RNA 指导的内切酶 Cas9 和 Cpf1 的全基因组脱靶活性来证明 BLISS 的灵敏度，观察到 Cpf1 比 Cas9 具有更高的特异性。我们的结果确立了 BLISS 作为一种通用、敏感和高效的方法，可用于许多应用中的全基因组 DSB 作图。
