Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System

By Baohui Chen, Luke A. Gilbert, Beth A. Cimini, J. Schnitzbauer, Wei Zhang, Gene‐Wei Li, Jason Park, Elizabeth H Blackburn, Jonathan S. Weissman, Lei S. Qi and 1 more. Cell, 2013

1,916 citations recorded by the source corpus.

SUMMARY The spatiotemporal organization and dynamics of chromatin play critical roles in regulating genome function. However, visualizing specific, endogenous genomic loci remains challenging in living cells. Here, we demonstrate such an imaging technique by repurposing the bacterial CRISPR/Cas system. Using an EGFP-tagged endonuclease-deficient Cas9 protein and a structurally optimized small guide (sg) RNA, we show robust imaging of repetitive elements in telomeres and coding genes in living cells. Furthermore, an array of sgRNAs tiling along the target locus enables the visualization of non-repetitive genomic sequences. Using this method, we have studied telomere dynamics during elongation or disruption, the subnuclear localization of the MUC4 loci, the cohesion of replicated MUC4 loci on sister chromatids, and their dynamic behaviors during mitosis. This CRISPR imaging tool has potential to significantly improve the capacity to study the conformation and dynamics of native chromosomes in living human cells.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/12e199b394000955acee01f2c3c8c3f634c1b319

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Citations in the corpus

Target specificity of the CRISPR-Cas9 system (2014, positive): “To enable site-specific labeling and imaging of endogenous loci in living cells, GFP has also been fused to dCas9 [31].”
Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems (2016, positive): “The dCas9 has been used for transcriptional activation and repression (135–138), localizing fluorescent protein labels (139), and recruiting histone-modifying enzymes (140, 141).”
The CRISPR/Cas9 system for plant genome editing and beyond. (2015, positive): “For example, dCas9 has been fused with a fluorescent protein to visualize selected loci in living cells, providing a useful tool for studying chromosome structure and dynamics (Anton et al., 2014; Chen et al., 2013).”
Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery (2014, methodology): “The DNA template encoding for a T7 promoter, a 20 nt target sequence and an optimized sgRNA scaffold (Chen et al., 2013) was assembled from synthetic oligonucleotides (Integrated DNA technologies, San Diego, CA) by overlapping PCR. Briefly, for the EMX1 sgRNA template, the PCR reaction contains 20…”
CRISPR/Cas Genome Editing and Precision Plant Breeding in Agriculture. (2019, positive): “dCas9 fused with a fluorescent protein, such as GFP, can be used to visualize DNA loci harboring repetitive sequences and to label endogenous centromeres, pericentric regions, and telomeres with single or multiplex sgRNAs (16) (Figure 2c).”
A CRISPR Dropout Screen Identifies Genetic Vulnerabilities and Therapeutic Targets in Acute Myeloid Leukemia (2016, positive): “To increase CRISPR-Cas9 efficiency, we first tested a gRNA scaffold optimized for CRISPR imaging (Chen et al., 2013) and found that, consistent with the results shown in a recent report (Dang et al.”
Development and Applications of CRISPR-Cas9 for Genome Engineering (2014, neutral): “Fluorescently tagged Cas9 labeling of specific DNA loci was recently developed as a powerful live-cell-imaging alternative to DNAFISH (Chen et al., 2013) (Figure 6H).”