CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells

Wulan Deng, Xinghua Shi, Robert Tjian, Timothée Lionnet, Robert H. Singer

Research output: Contribution to journalArticle

86 Citations (Scopus)

Abstract

Direct visualization of genomic loci in the 3D nucleus is important for understanding the spatial organization of the genome and its association with gene expression. Various DNA FISH methods have been developed in the past decades, all involving denaturing dsDNA and hybridizing fluorescent nucleic acid probes. Here we report a novel approach that uses in vitro constituted nuclease-deficient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated caspase 9 (Cas9) complexes as probes to label sequence-specific genomic loci fluorescently without global DNA denaturation (Cas9-mediated fluorescence in situ hybridization, CASFISH). Using fluorescently labeled nuclease-deficient Cas9 (dCas9) protein assembled with various single-guide RNA (sgRNA), we demonstrated rapid and robust labeling of repetitive DNA elements in pericentromere, centromere, G-rich telomere, and coding gene loci. Assembling dCas9 with an array of sgRNAs tiling arbitrary target loci, we were able to visualize nonrepetitive genomic sequences. The dCas9/sgRNA binary complex is stable and binds its target DNA with high affinity, allowing sequential or simultaneous probing of multiple targets. CASFISH assays using differently colored dCas9/sgRNA complexes allow multicolor labeling of target loci in cells. In addition, the CASFISH assay is remarkably rapid under optimal conditions and is applicable for detection in primary tissue sections. This rapid, robust, less disruptive, and cost-effective technology adds a valuable tool for basic research and genetic diagnosis.

Original languageEnglish (US)
Pages (from-to)11870-11875
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume112
Issue number38
DOIs
StatePublished - Sep 22 2015

Fingerprint

Clustered Regularly Interspaced Short Palindromic Repeats
Guide RNA
Caspase 9
Fluorescence In Situ Hybridization
DNA
Nucleic Acid Probes
Nucleic Acid Denaturation
Genetic Research
Centromere
Telomere
Genome
Technology
Gene Expression
Costs and Cost Analysis
Genes
Proteins

Keywords

  • Cas9
  • CRISPR
  • FISH
  • Genome organization
  • Halo

ASJC Scopus subject areas

  • General

Cite this

CASFISH : CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells. / Deng, Wulan; Shi, Xinghua; Tjian, Robert; Lionnet, Timothée; Singer, Robert H.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 112, No. 38, 22.09.2015, p. 11870-11875.

Research output: Contribution to journalArticle

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