4.1 Article

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

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STAR PROTOCOLS
卷 3, 期 4, 页码 -

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ELSEVIER
DOI: 10.1016/j.xpro.2022.101734

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资金

  1. Canadian Institutes of Health Research (CIHR)
  2. CIHR CGS
  3. Ontario Graduate Scholarship (OGS)
  4. Ruggles Innovation Award
  5. Adel S. Sedra Award
  6. CIHR [388041, 399687]
  7. Canada Research Chairs Program (CRC) [950-230661]
  8. Ontario Ministry of Research and Innovation (MRI-ERA) [ER13-09-111]

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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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