[16]. Additionally, this method requires the expression of the
DNA-binding LacI repressor protein fused with a fluorescent protein, such as GFP or variants (CFP, YFP, RFP, etc.). The expression
of these proteins should be somewhat weak in order to avoid the
background fluorescence masking the specific signal at the tagged
sequence. The tagged sequence is detected through the binding of
the repressor protein (LacI-GFP) to the lacO repeats, allowing the
tracking of specific chromosomal sites by in vivo fluorescence
microscopy. The CRISPR imaging system is based on a catalytically
inactive Cas9, fused to a fluorescent protein, where guide RNAs are
designed for a specific sequence in the genome allowing the dCas9
to recognize and tag the locus of interest [17, 18]. These genetagging techniques have been extensively used in organisms such as
yeast or mammalian cells, both to characterize chromatin mobility
during DNA damage and to study mobility of actively transcribed
loci [9, 13, 19–22]. In Arabidopsis thaliana, the lacO/LacI system
has been used in order to study chromosome organization [23],
changes in nuclear positioning of Polycomb targets [24], chromatin dynamics in endoreplicated pavement cells [25], as well as
alterations of lacO positioning upon DNA damage [26]. One drawback of the lacO/LacI system is that the LacO repeats tend to form
heterochromatin, which silences the locus and sometimes the
neighboring genes [27]. To overcome this issue LacO repeats can
be interspaced with random short sequences (~10mers), which
were shown to reduce problems with repeat-induced silencing
[24, 28]. Unlike lacO/LacI, the CRISPR imaging system has the
advantage that it recognizes the endogenous sequence in the
genome and is less likely to perturb the underlying chromatin
structure. In plants, telomere mobility has been measured using
the CRISPR imaging system in Nicotiana benthamiana [18]; however this method has not yet been successfully applied in Arabidopsis thaliana. Indeed, and despite the great progress made in the
CRISPR-based imaging, many challenges still remain to be settled
before this method is readily available for use in all systems, and in
particular, issues associated with imaging of non-repetitive
sequences and off-target binding need to yet be overcome [29].
In both systems, the tagged sequence is seen as a bright spot in
the nucleus, which can be followed by in vivo time-lapse imaging.
The mobility and trajectory of this bright dot can be analyzed to
obtain information regarding the mechanisms underlying its movement. However, the simple tracking of the tagged loci will inform
only on changes in position as a function of time, which is not
enough to characterize loci mobility behavior. To this end, one
method typically used to extract information about a moving particle tracking data is mean square displacement (MSD) analysis [30].
In this chapter, we will describe the protocol we use to visualize
and quantify chromatin movement using the lacO/LacI genetagging system, particularly focusing in Arabidopsis roots, but a
214
Anis Meschichi and Stefanie Rosa
DNA-binding LacI repressor protein fused with a fluorescent protein, such as GFP or variants (CFP, YFP, RFP, etc.). The expression
of these proteins should be somewhat weak in order to avoid the
background fluorescence masking the specific signal at the tagged
sequence. The tagged sequence is detected through the binding of
the repressor protein (LacI-GFP) to the lacO repeats, allowing the
tracking of specific chromosomal sites by in vivo fluorescence
microscopy. The CRISPR imaging system is based on a catalytically
inactive Cas9, fused to a fluorescent protein, where guide RNAs are
designed for a specific sequence in the genome allowing the dCas9
to recognize and tag the locus of interest [17, 18]. These genetagging techniques have been extensively used in organisms such as
yeast or mammalian cells, both to characterize chromatin mobility
during DNA damage and to study mobility of actively transcribed
loci [9, 13, 19–22]. In Arabidopsis thaliana, the lacO/LacI system
has been used in order to study chromosome organization [23],
changes in nuclear positioning of Polycomb targets [24], chromatin dynamics in endoreplicated pavement cells [25], as well as
alterations of lacO positioning upon DNA damage [26]. One drawback of the lacO/LacI system is that the LacO repeats tend to form
heterochromatin, which silences the locus and sometimes the
neighboring genes [27]. To overcome this issue LacO repeats can
be interspaced with random short sequences (~10mers), which
were shown to reduce problems with repeat-induced silencing
[24, 28]. Unlike lacO/LacI, the CRISPR imaging system has the
advantage that it recognizes the endogenous sequence in the
genome and is less likely to perturb the underlying chromatin
structure. In plants, telomere mobility has been measured using
the CRISPR imaging system in Nicotiana benthamiana [18]; however this method has not yet been successfully applied in Arabidopsis thaliana. Indeed, and despite the great progress made in the
CRISPR-based imaging, many challenges still remain to be settled
before this method is readily available for use in all systems, and in
particular, issues associated with imaging of non-repetitive
sequences and off-target binding need to yet be overcome [29].
In both systems, the tagged sequence is seen as a bright spot in
the nucleus, which can be followed by in vivo time-lapse imaging.
The mobility and trajectory of this bright dot can be analyzed to
obtain information regarding the mechanisms underlying its movement. However, the simple tracking of the tagged loci will inform
only on changes in position as a function of time, which is not
enough to characterize loci mobility behavior. To this end, one
method typically used to extract information about a moving particle tracking data is mean square displacement (MSD) analysis [30].
In this chapter, we will describe the protocol we use to visualize
and quantify chromatin movement using the lacO/LacI genetagging system, particularly focusing in Arabidopsis roots, but a
214
Anis Meschichi and Stefanie Rosa