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

Negin Khosraviani; Karan J Abraham; Janet N Y Chan; Karim Mekhail

doi:10.1016/j.xpro.2022.101734

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

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

Authors

Negin Khosraviani¹, Karan J Abraham¹, Janet N Y Chan², Karim Mekhail³

Affiliations

¹ Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, 661 University Avenue, Toronto, ON, Canada.
² Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, 661 University Avenue, Toronto, ON, Canada. Electronic address: janetny.chan@utoronto.ca.
³ Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, 661 University Avenue, Toronto, ON, Canada; Canada Research Chairs Program, Faculty of Medicine, University of Toronto, 1 King's College Circle, Toronto, ON, Canada. Electronic address: karim.mekhail@utoronto.ca.

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).

Keywords: CRISPR; Cell biology; Cell culture; Gene expression; Genetics; Molecular biology.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Clustered Regularly Interspaced Short Palindromic Repeats*
DNA / metabolism
Genome
R-Loop Structures*
RNA / genetics

Substances

RNA
DNA

Abstract

Publication types

MeSH terms

Substances

Grants and funding