基于 RNaseH1 的 CRISPR 调控基因座相关 R 环的方案。

Protocol to use RNaseH1-based CRISPR to modulate locus-associated R-loops.

机构信息

Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, 661 University Avenue, Toronto, ON, Canada.

出版信息

STAR Protoc. 2022 Dec 16;3(4):101734. doi: 10.1016/j.xpro.2022.101734. Epub 2022 Sep 29.

DOI:10.1016/j.xpro.2022.101734

PMID:36178790

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9525952/

Abstract

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).

摘要

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)。