Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA.
Department of Molecular and Cellular Biology, Harvard
University, Cambridge, MA, USA.
Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA.
Pathology, Beth Israel Deaconess Medical Center, Boston, MA, USA. Present addresses:
Department of Pharmacology, The University of Washington,
Seattle, WA, USA.
Department of Biochemistry, University of Colorado, BioFrontiers, Boulder, CO, USA. *e-mail: email@example.com;
he 3D organization of DNA is critical in the establishment of
cellular states and is frequently dysregulated in disease
organization relies on regulatory aspects, such as nonrandom
chromosomal positioning, chromosomal substructures and proper
ties, and ultimately the position of a given locus in the nucleus
Underscoring the importance of these processes, several recent
studies have revealed mechanistic links between chromosomal
topology and disease. For example, structural chromosomal aber
rations can result in pathogenic rewiring of enhancer–promoter
This raises the question of how heterozygous structural aber
rations affect nuclear organization properties. Addressing this has
been difficult for several reasons. (1) An allele-specific imaging
approach is required, but so far, there are only two allele-specific
techniques for fixed cells: using copy number variations and oli
. (2) The Hi-C method has been adopted to study
molecular interactions of haplotype genomes but lacks the ability
to assess spatiotemporal dimensions
. (3) These techniques all
use cross-linking to fix specimens, which prevents the elucidation of
spatiotemporal dynamics. Such fixative treatments can also disrupt
higher-order nuclear architecture and cause nonphysiological arti
. In sum, to date, the real-time study of specific allele position-
ing in living cells has been infeasible.
To achieve allele-specific resolution, properties, and dynam
ics of gene loci, we took advantage of the programmable ability of
the CRISPR–Cas9 system to recognize specific DNA sequences to
engineer a live-cell imaging technique that is able to resolve indi
vidual alleles in order to study their properties in living cells. Using
this technique, termed single nucleotide polymorphism CRISPR
live-cell imaging (SNP-CLING), we found that it is a highly spe
cific and universally applicable method that can resolve allelic
positioning relative to nuclear subcompartments (for example, the
nucleolus) and allele-specific interactions between nonhomolo
gous chromosomes. Importantly, all of these factors can be inves-
tigated in living cells.
We applied SNP-CLING to two long noncoding RNA (lncRNA)
loci, Firre and CISTR-ACT, which are involved in heterozygous struc
tural aberrations that cause Mendelian disease
encompassing FIRRE have been found in patients with periventric
ular nodular heterotopia with polymicrogyria
, and translocations
of CISTR-ACT are causally associated with brachydactyly
methods cannot resolve the resulting implications of these hetero
zygous aberrations on higher-order nuclear architecture.
Here, we first validate the specificity and accuracy of SNP-
CLING and explore allelic positioning across space and time. Using
3D imaging, we determined that alleles stably maintain similar
positions close to the nucleolus, although each studied locus occu
pied a unique localization within the nucleus. Next, we extended
our analysis and performed allele-specific imaging across time
(4D) to elucidate spatiotemporal allele positioning in relation to
the major subnuclear compartment of the nucleolus. We found that
alleles are stably positioned through time in human and mouse
cells. This finding suggests that not only are chromosome territories
stably positioned, but also, specific spatial distances are maintained
between alleles or loci. Moreover, through time, these distances are
preserved, suggesting that there is no random movement of alleles
relative to each other or relative to nuclear substructures such as
the nucleolus. Altogether, SNP-CLING is broadly applicable in deci
phering a multitude of previously intractable questions on chroma-
tin biology and nuclear architecture in living cells.
Implementing allele-specific SNP-CLING. To visualize each allele
of a locus simultaneously in a living cell, we leveraged a nuclease-
null mutant of the Streptococcus pyogenes Cas9 protein (dCas9) with
pools of two to three single-guide RNAs (sgRNAs) targeting each
Spatiotemporal allele organization by allele-
specific CRISPR live-cell imaging (SNP-CLING)
Philipp G. Maass
*, A. Rasim Barutcu
, David M. Shechner
, Catherine L. Weiner
and John L. Rinn
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 comprehen-
sive 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.
NATURE STRUCTURAL & MOLECULAR BIOLOGY | VOL 25 | FEBRUARY 2018 | 176–184 | www.nature.com/nsmb
© 2018 Nature America Inc., part of Springer Nature. All rights reserved.