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Related Experiment Video

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Laser Microdissection-Based Protocol for the LC-MS/MS Analysis of the Proteomic Profile of Neuromelanin Granules
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Adapting dCas9-APEX2 for subnuclear proteomic profiling.

Xin D Gao1, Tomás C Rodríguez1, Erik J Sontheimer1

  • 1RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, United States.

Methods in Enzymology
|January 30, 2019
PubMed
Summary
This summary is machine-generated.

This study introduces a CRISPR-Cas9 method to map DNA-binding proteins at specific genome locations. This technique aids in understanding genome organization and nuclear protein functions.

Keywords:
APEXChromosomeMass spectrometryProteomedCas9

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Area of Science:

  • Molecular Biology
  • Genomics
  • Proteomics

Background:

  • Genome organization and subnuclear protein localization are crucial for cellular functions like gene expression and DNA replication.
  • Advanced techniques like chromatin conformation capture have improved understanding of genome architecture.
  • Profiling DNA-associated proteins at specific sites is vital for detailed genomic studies.

Purpose of the Study:

  • To describe the dCas9-APEX2 experimental approach for site-specific profiling of DNA-associated proteins.
  • To enable high-throughput and unbiased analysis of proteomic landscapes at various genomic loci.
  • To facilitate the understanding of nuclear and chromosome biology through detailed protein localization.

Main Methods:

  • Utilizing the RNA-programmable CRISPR-Cas9 platform combined with an enzymatic labeling system (APEX2).
  • Site-specifically targeting DNA sequences to enzymatically label local proteins with biotin.
  • Quantitatively analyzing the biotinylated proteome to identify DNA-associated proteins.

Main Results:

  • Demonstrated the dCas9-APEX2 method for high-resolution proteomic profiling at specific DNA loci.
  • Enabled unbiased identification of proteins associated with both repetitive and nonrepetitive genomic regions.
  • Provided a powerful tool for studying genome organization and protein localization.

Conclusions:

  • The dCas9-APEX2 approach offers unprecedented ease and resolution for mapping DNA-associated proteins.
  • This method significantly advances the study of nuclear and chromosome biology.
  • Optimization and extension of this pipeline will further enhance its utility in genomic research.