CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells.
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.
Transcription Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147; dengw@janelia.hhmi.org singerr@janelia.hhmi.org.
Wulan
W
Deng
Transcription Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147;
Xinghua
X
Shi
Transcription Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147; Department of Molecular and Cell Biology, University of California, Berkeley, CA 94707;
Robert
R
Tjian
Transcription Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147;
Timothée
T
Lionnet
Transcription Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147; Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461; Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY 10461 dengw@janelia.hhmi.org singerr@janelia.hhmi.org.
Robert H
RH
Singer
D000818:Animals; D001665:Binding Sites; D064130:CRISPR-Associated Proteins / Q000378:metabolism*; D002232:Carbocyanines / Q000378:metabolism; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D003116:Color; D004247:DNA / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D056426:Genetic Loci; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D000094704:RNA, Guide, CRISPR-Cas Systems; D013194:Staining and Labeling
D064130:CRISPR-Associated Proteins; D002232:Carbocyanines; D005456:Fluorescent Dyes; D000094704:RNA, Guide, CRISPR-Cas Systems; C085321:cyanine dye 5; D004247:DNA
CRISPR; Cas9; FISH; Halo; genome organization