期刊
STAR PROTOCOLS
卷 3, 期 4, 页码 -出版社
ELSEVIER
DOI: 10.1016/j.xpro.2022.101734
关键词
-
资金
- Canadian Institutes of Health Research (CIHR)
- CIHR CGS
- Ontario Graduate Scholarship (OGS)
- Ruggles Innovation Award
- Adel S. Sedra Award
- CIHR [388041, 399687]
- Canada Research Chairs Program (CRC) [950-230661]
- Ontario Ministry of Research and Innovation (MRI-ERA) [ER13-09-111]
This study presents a locus-associated R-loop-modulating system that can attribute functions to R-loops in a locus-specific manner using an inducible RNaseH1-EGFP-dCas9 chimaera. The study confirms the expression, localization, locus-targeted association, and modulation of R-loops in cis or trans using immunoblotting, microscopy, and chromatin and DNA-RNA immunoprecipitation.
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).
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