A technique to detect native R-loops genome-wide without immunoprecipitation

We adapted the nuclease-targeting approach CUT&RUN (Skene & Henikoff, eLife 2017) to map R-loops in vivo. We show that this approach accurately identifies R-loops that are sensitive to both Actinomycin D and RNase H and largely overlap with previously identified regions containing R-loops. We also show that MapR can identify R-loops genome-wide from much smaller input material than existing techniques. DNA fragments from R-loop were relased by incubating nuclei with GST-RNAseH∆-Mnase in vitro (MapR), where ∆ indicates a mutation that kills the catalytic activity of RNase H. As a control, we incubated the nuclei with GST-Mnase lacking the RNase H moiety (MapR control). We also compared these results with those obtained by expressing a catalytically inactive RNase H fused to the FLAG epitope tag directly in cells and then performing a more conventional CUT&RUN assay (Skene & Henikoff, eLife 2017) using an anti-FLAG antibody. In that case the control is performing the CUT&RUN assay with a non-specific IgG control.

我们对靶向核酸酶的CUT&RUN技术（Skene与Henikoff，《eLife》2017）进行了适配，以在体内绘制R环（R-loops）的图谱。我们证实，该方法可精准识别对放线菌素D（Actinomycin D）与核糖核酸酶H（RNase H）均敏感的R环，且这些R环在很大程度上与此前已报道的含R环区域相重合。我们还证实，相较于现有技术，MapR可从更少的起始实验材料中实现全基因组范围的R环鉴定。具体而言，我们通过将细胞核与GST-RNAseHΔ-Mnase体外共孵育，释放R环对应的DNA片段（即MapR实验），其中Δ代表一种可使核糖核酸酶H（RNase H）催化活性丧失的突变。作为对照，我们将细胞核与缺失核糖核酸酶H结构域的谷胱甘肽S-转移酶（GST）-微球菌核酸酶（Mnase）共孵育（即MapR对照）。我们还将上述结果与直接在细胞中表达融合FLAG标签的催化失活型核糖核酸酶H，随后使用抗FLAG抗体开展更常规的CUT&RUN实验（Skene与Henikoff，《eLife》2017）所得到的结果进行了比较。在此情况下，对照实验为使用非特异性IgG抗体开展CUT&RUN实验。