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.
journal_name
Proc Natl Acad Sci U S Aauthors
Deng W,Shi X,Tjian R,Lionnet T,Singer RHdoi
10.1073/pnas.1515692112subject
Has Abstractpub_date
2015-09-22 00:00:00pages
11870-5issue
38eissn
0027-8424issn
1091-6490pii
1515692112journal_volume
112pub_type
杂志文章abstract::The binding of 125I-labeled wheat germ agglutinin (WGA) to parental and three distinct WGA-resistant Chinese hamster ovary cell lines possessing modified cell surface carbohydrate structures has been examined over a 10(6)-fold range of WGA concentrations. The Scatchard plot for WGA binding to parental cells was comple...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.74.11.5056
更新日期:1977-11-01 00:00:00
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
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更新日期:2011-03-29 00:00:00
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
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更新日期:2008-01-15 00:00:00
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
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更新日期:2007-02-13 00:00:00
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
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更新日期:1970-04-01 00:00:00