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

Negin Khosraviani1, Karan J Abraham1, Janet N Y Chan1

  • 1Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, 661 University Avenue, Toronto, ON, Canada.

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|September 30, 2022
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Summary

This study introduces LasR, a system for locus-associated R-loop modulation, enabling researchers to investigate R-loop functions genome-wide. This method allows precise control and analysis of R-loop structures at specific genomic locations.

Keywords:
CRISPRCell biologyCell cultureGene expressionGeneticsMolecular biology

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Area of Science:

  • Molecular Biology
  • Genetics
  • Genomics

Background:

  • R-loops are nucleic acid structures with significant genomic roles.
  • Current methods lack locus-specific R-loop analysis capabilities.
  • Understanding locus-associated R-loop functions is crucial for genome regulation studies.

Purpose of the Study:

  • To present a novel system, LasR, for locus-associated R-loop modulation.
  • To enable precise investigation of R-loop functions at specific genomic sites.
  • To provide a detailed protocol for utilizing this system.

Main Methods:

  • Design of single-guide RNAs (sgRNAs) using in silico tools.
  • Transfection of cells with an inducible RNaseH1-EGFP-dCas9 chimaera and sgRNAs.
  • Confirmation of protein expression, localization, and target association via immunoblotting and microscopy.
  • Assessment of R-loop modulation using chromatin and DNA-RNA immunoprecipitation.

Main Results:

  • Successful development and implementation of the LasR system.
  • Demonstration of locus-targeted association of the RNaseH1-EGFP-dCas9 chimaera.
  • Verification of R-loop modulation in cis and trans at targeted loci.
  • Validation of the protocol's efficacy in studying R-loop dynamics.

Conclusions:

  • The LasR system offers a powerful tool for locus-specific R-loop modulation.
  • This protocol facilitates the study of R-loop functions across the genome.
  • Enables new insights into the roles of R-loops in gene regulation and genome stability.