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Nat Struct Mol Biol. 2018 Feb;25(2):176-184. doi: 10.1038/s41594-017-0015-3. Epub 2018 Jan 8.

Spatiotemporal allele organization by allele-specific CRISPR live-cell imaging (SNP-CLING).

Maass PG1, Barutcu AR2,3, Shechner DM2,4,3,5, Weiner CL2,4,3, Melé M2,3, Rinn JL6,7,8,9,10.

Author information

1
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA. philippmaass@fas.harvard.edu.
2
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA.
3
Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA.
4
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA.
5
Department of Pharmacology, The University of Washington, Seattle, WA, USA.
6
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA. john.rinn@colorado.edu.
7
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA. john.rinn@colorado.edu.
8
Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. john.rinn@colorado.edu.
9
Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA, USA. john.rinn@colorado.edu.
10
Department of Biochemistry, University of Colorado, BioFrontiers, Boulder, CO, USA. john.rinn@colorado.edu.

Abstract

Imaging and chromatin capture techniques have provided important insights into our understanding of nuclear organization. A limitation of these techniques is the inability to resolve allele-specific spatiotemporal properties of genomic loci in living cells. Here, we describe an allele-specific CRISPR live-cell DNA imaging technique (SNP-CLING) to provide the first comprehensive insights into allelic positioning across space and time in mouse embryonic stem cells and fibroblasts. With 3D imaging, we studied alleles on different chromosomes in relation to one another and relative to nuclear substructures such as the nucleolus. We find that alleles maintain similar positions relative to each other and the nucleolus; however, loci occupy unique positions. To monitor spatiotemporal dynamics by SNP-CLING, we performed 4D imaging and determined that alleles are either stably positioned or fluctuating during cell state transitions, such as apoptosis. SNP-CLING is a universally applicable technique that enables the dissection of allele-specific spatiotemporal genome organization in live cells.

PMID:
29343869
PMCID:
PMC5805655
DOI:
10.1038/s41594-017-0015-3
[Indexed for MEDLINE]
Free PMC Article

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