Dear Editor
Visualization of chromosome shapes and dynamics in a live cell is highly desirable
and necessary in many areas of cell biology. For example, the copy number of a particular
chromosome in cancer cells is often abnormal (e.g., more than two), and therefore
probing chromosome copy numbers can aid cancer diagnosis. During interphase, each
chromosome exists in its own territory in the nucleus, which can be imaged by fluorescence
in situ hybridization (FISH) using sequence-specific probes of different colors
1,2
. However, such chromosome painting has only been possible in fixed cells, and is
not suitable for dynamic monitoring of live cells. Therefore, it would be valuable
to visualize DNA replication of one chromosome during interphase, and follow chromosome
dynamics in the M phase.
Recent development of clustered regularly interspaced short palindromic repeats/CRISPR-associated
proteins (CRISPR/Cas)
3
has provided a powerful tool for live-cell imaging of genomic loci
4
. In particular, the nuclease defective Cas9 (dCas9) fused with enhanced green fluorescent
protein (EGFP) is used to target a particular DNA sequence upstream of a protospacer
adjacent motif (PAM) sequence. Such targeting is achieved through Watson-Crick base
pairing of ∼20-bp single-guide RNA (sgRNA) that is pre-complexed with the dCas9-EGFP
protein. The targeted loci can thus be fluorescently labeled in live mammalian cells
5,6,7,8
. However, the labeling achieved by this method is usually restricted to the genomic
loci that consist of repetitive sequences, and has not been attempted to track an
entire chromosome in a live cell.
Here we report the specific labeling of a large number of loci in the genome, which
makes it possible to paint an entire chromosome in a live cell. To do so, we designed
a new strategy using a large number of sgRNAs targeting mainly the non-repetitive
regions of the chromosome (Figure 1A and Supplementary information, Figure S1A). To
design sgRNAs, we scanned the sequence of the entire chromosome 9 on human reference
genome hg19. Each 19-23 bp genome sequence upstream of a PAM sequence NGG was taken
as a candidate target region. Because the efficiency of sgRNA binding is dependent
on its GC content
9,10
, sgRNAs with GC content of 45%-65% were selected (Supplementary information, Figure
S1B). The sgRNAs that could also bind to other chromosomes were removed in order to
assure labeling specificity and reduce fluorescent background. Additionally, when
multiple targeting sequences overlapped in a chromosome region, only one of them was
selected. Among all sgRNAs, only one protein-coding gene (DNAJB5) contains sgRNA-binding
sites in the exon region (Supplementary information, Figure S1C), but its expression
level was not affected by lentivirus infection or dCas9-EGFP/sgRNA targeting (Supplementary
information, Figure S1D).
To obtain high densities of sgRNA that would give signals significantly above the
intracellular fluorescent background, especially when the chromatin is in open state
during G and S phases, we chose 15 clusters of target sites (c1-c15) on chromosome
9, each spanning 5 Kb and containing more than 30 targets. The clusters are placed
at least 5 Mbp away from each other (Figure 1A). In total, we selected 1 124 sgRNAs
that are distributed on chromosome 9 (Supplementary information, Table S1).
As shown in Supplementary information, Figure S1A, a cell line stably expressing dCas9-EGFP
was constructed from one HeLa cell by lentiviral infection. In the meantime, the 1
124 sgRNAs were packaged into lentiviruses and used to infect HeLa cells expressing
dCas9-EGFP for three times. After labeling the chromosome with the 1 124 sgRNAs, we
imaged cells in the S phase using a Nikon structured illumination microscope (N-SIM)
equipped with a 100× TIRF oil immersion objective (NA 1.49). Figure 1B shows that
the projected z-stack EGFP images. We attribute the three bright EGFP signal regions
in the nucleus to three copies of chromosome 9, which is consistent with the karyotype
of the HeLa cells we used (Supplementary information, Figure S1E). We noted that the
EGFP signal is stronger in the S phase, when DNA replication takes place, than in
the G1 phase, indicating that dCas9-EGFP can bind rapidly to newly synthesized DNA.
Taken together, the imaging results suggest the efficacy of our method in visualizing
a desired chromosomal territory in a live cell.
Figure 1C shows a projected z-stack of N-SIM fluorescence images taken in the prophase
during mitosis. The high resolution of structured illumination microscopy and the
strong signal due to chromosome condensation, allowed for the visualization of the
three pairs of sister chromatids (Figure 1C) after DNA replication.
We next aimed to verify the dCas9-EGFP signals with FISH using probes targeting repetitive
sequences so that a single FISH probe could access many sites. We used another two
sgRNA sequences, C9-1 and C9-2, which were previously described and named
7
, to replace the three non-repetitive clusters c13-c15. C9-1 binds to a region with
pericentromeric repeats on chromosome 9 and C9-2 targets 115 sites within a 5 Kb region.
We delivered 800 sgRNAs from c1-c12 clusters as well as C9-1 and C9-2 (802 sgRNAs
in total) into the HeLa cells expressing dCas9-EGFP by lentivirus infection (Supplementary
information, Figure S2A). Before painting the entire chromosome, we confirmed that
each of the 12 clusters of sgRNAs could individually label the corresponding genomic
locus efficiently (Supplementary information, Figure S2B). As expected, we observed
the co-localization of dCas9-EGFP and FISH labeling of C9-1 and C9-2 (Supplementary
information, Figure S2C).
After labeling the chromosome with the 802 sgRNAs, we found the imaging results for
the chromosome territory and three pairs of sister chromatids are very similar to
those labeled with c1-c15 containing 1 124 sgRNAs (Supplementary information, Figure
S2D and E). The labeled chromosome 9 in the M phase can be easily seen even with a
wide-field fluorescence microscopy. Figure 1D shows the cells in the prophase, metaphase,
anaphase and telophase during mitosis. The cells were also stained with a DNA-specific
dye Hoechst 33342, which allows co-localization of chromosome 9 with the rest of chromosomes.
The three pairs of sister chromatids in the prophase and metaphase, though not clearly
resolved with this reduced resolution, split to two sets of separate chromosomes in
the anaphase and telophase. As a final control experiment, Figure 1E shows co-localization
of labeled chromosome 9 of the EGFP signal with the Cy3-tagged C9-1 and C9-2 FISH
probes. The fact that the EGFP areas were larger than the diffraction-limited FISH
areas proves again the successful labeling of chromosome 9.
Having demonstrated stable labeling of chromosome 9, we next applied our method to
dynamic monitoring using a DeltaVision Imaging System (Applied Precision/GE) equipped
with a 100×/1.4 NA oil immersion objective, which allows less photobleaching, longer
time for data acquisition, and confocality based on deconvolution. Figure 1F and Supplementary
information, Movie S1 show chromosome dynamics at a single chromosome level for a
cell in the S phase. Supplementary information, Figure S2F and Movie S2 show the dynamics
of chromosome 9 during a period of 2 h from the late S phase to M phase. In addition
to the three chromosome 9 spots, the nucleoli exhibit unintended EGFP signal, likely
due to nonspecific binding of dCas9-EGFP protein with small RNAs in the nucleoli
8
. During the data collection period, EGFP signal from the nucleoli faded away due
to the disappearance of the nucleoli in the M phase, while chromosome 9 fluorescent
signal became stronger due to chromosome condensation. Supplementary information,
Movie S3 shows another cell in the M phase for a period of ∼3 h. After cell division,
the two daughter cells maintained strong fluorescence signals from the replicated
chromosomes, indicating that chromosome labeling is kept in the daughter cells. In
this movie, significant conformational fluctuation of the condensed chromosomes was
observed. Although hundreds of sgRNAs were delivered by lentivirus into the cells,
no obvious effects on cell proliferation were observed (Supplementary information,
Figure S2G).
To evaluate how many sgRNAs were actually introduced to each cell, we carried out
DNA sequencing of clonally amplified cells after PCR amplification with the common
primers for sgRNA sequences
11
. Figure 1G shows the distribution of the detected 510 non-repetitive sgRNA sequences
in chromosome 9 in one clonally amplified population. Single clones with higher labeling
efficiency (S1) had more sgRNAs sequences incorporated than single clones with weaker
fluorescence (S34) (Supplementary information, Figure S2H). Figure 1H shows the distribution
of read numbers for all sgRNA sequences in the population of cells, showing that majority
of the sgRNAs has been sufficiently incorporated into the cell population.
To address the question of how many sgRNAs are sufficient for painting a chromosome,
we reduced the number of non-repetitive sgRNAs to 485. We found no significant deterioration
of the image quality when compared with the images produced with 802 sgRNAs (Supplementary
information, Figure S2I), when we imaged chromosome 9 at different phases of the M
phase. Based on this, we conclude that more than 20 sgRNAs in each cluster and at
least 300 types of total sgRNAs are needed in one cell for effective chromosome 9
painting. As the required number of the sgRNAs should be dependent on the length of
the chromosome, we anticipate that using our design strategy ∼800 sgRNAs should be
enough to label the longest human chromosome 1, which is about twice as long as chromosome
9, while ∼100 sgRNAs could be enough for the shortest chromosome. Of course, for painting
other chromosomes, additional optimization and validation may be required.
In summary, by introducing hundreds of specific and non-repetitive sgRNAs in a human
cell, we are able to paint an entire chromosome in a live cell for fluorescent imaging.
We have visualized the spatial arrangements of homologous chromosomes and sister chromatids
and tracked the movement of a particular chromosome in dividing cells. Our method
will facilitate studies of functional organization of chromosomes, interactions among
different chromosome regions, and long-term chromosomal dynamics in live mammalian
cells.