crispr-mediated-reorganization-of-chromatin-loop-structure

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Stanford University School of Medicine

Protein glycosylation is of increasing interest due to its important roles in protein function and aberrant expression with disease. Characterizing protein glycosylation remains analytically challenging due to its low abundance, ion suppression issues, and microheterogeneity at glycosylation sites, especially in complex samples such as human plasma. In this study, the utility of three common N-linked glycopeptide enrichment techniques is compared using human plasma. By analysis on an LTQ-Orbitrap Elite mass spectrometer, electrostatic repulsion hydrophilic interaction liquid chromatography using strong anion exchange solid-phase extraction (SAX-ERLIC) provided the most extensive N-linked glycopeptide enrichment when compared with multilectin affinity chromatography (M-LAC) and Sepharose-HILIC enrichments. SAX-ERLIC enrichment yielded 191 unique glycoforms across 72 glycosylation sites from 48 glycoproteins, which is more than double that detected using other enrichment techniques. The greatest glycoform diversity was observed in SAX-ERLIC enrichment, with no apparent bias toward specific glycan types. SAX-ERLIC enrichments were additionally analyzed by an Orbitrap Fusion Lumos mass spectrometer to maximize glycopeptide identifications for a more comprehensive assessment of protein glycosylation. In these experiments, 829 unique glycoforms were identified across 208 glycosylation sites from 95 plasma glycoproteins, a significant improvement from the initial method comparison and one of the most extensive site-specific glycosylation analysis in immunodepleted human plasma to date. Data are available via ProteomeXchange with identifier PXD005655.

Parallel Comparison of N-Linked Glycopeptide Enrichment Techniques Reveals Extensive Glycoproteomic Analysis of Plasma Enabled by SAX-ERLIC.

Chromatin looping is key to gene regulation, yet no broadly applicable methods to selectively modify chromatin loops have been described. We have engineered a method for chromatin loop reorganization using CRISPR-dCas9 (CLOuD9) to selectively and reversibly establish chromatin loops. We demonstrate the power of this technology to selectively modulate gene expression at targeted loci.

Abel Bermudez

Canary Center for Cancer Early Detection,
Department of Radiology,
Canary Center for Cancer Early Detection, Department of Radiology

Stanford University School of Medicine

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Abel Bermudez

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Stanford University School of Medicine

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Canary Center for Cancer Early Detection,
Department of Radiology,
Canary Center for Cancer Early Detection, Department of Radiology