Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, 661 University Avenue, Toronto, ON, Canada.
Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, 661 University Avenue, Toronto, ON, Canada.
STAR Protoc. 2022 Dec 16;3(4):101734. doi: 10.1016/j.xpro.2022.101734. Epub 2022 Sep 29.
Modulating R-loop triplex nucleic acid structures reveals their roles across the genome. However, common approaches cannot ascribe functions to R-loops in a locus-associated manner. This protocol presents the use of a locus-associated R-loop-modulating system (dubbed LasR), which employs an inducible RNaseH1-EGFP-dCas9 chimaera. We detail the in silico design of sgRNAs and their transfection with the chimaera, and outline steps confirming RNaseH1-EGFP-dCas9 expression, localization, locus-targeted association, and R-loop modulation in cis or trans using immunoblotting, microscopy, and chromatin and DNA-RNA immunoprecipitation. For complete details on the use and execution of this protocol, please refer to Abraham et al. (2020).
调控 R 环三链体结构揭示了它们在整个基因组中的作用。然而，常见的方法无法以与基因座相关的方式将功能归因于 R 环。本方案介绍了一种与基因座相关的 R 环调节系统（称为 LasR）的使用，该系统采用诱导型 RNaseH1-EGFP-dCas9 嵌合体。我们详细介绍了 sgRNA 的计算机设计及其与嵌合体的转染，并概述了使用免疫印迹、显微镜以及染色质和 DNA-RNA 免疫沉淀来确认 RNaseH1-EGFP-dCas9 表达、定位、基因座靶向关联以及顺式或反式 R 环调节的步骤。有关该方案的使用和执行的完整详细信息，请参阅 Abraham 等人。(2020)。
