Live-cell imaging was boosted with the application of
GFP-fused chromatin proteins including histones, condensins,
and others [4]. Nonetheless, this method is unable to detect specific genomic loci. Later, fluorescent repressor/operator systems
were used to allow specific labeling of genomic regions in comparison to chromatin proteins [5]. In plants, this technique was applied
to A. thaliana to compare the ploidy level of guard cells and
elongated epidermal root cells [6]. However, this method is not
able to label predefined genomic regions as a random insertion of
the tandem operator repeats into the genome occurs after plant
transformation. In addition, methylation at operator insertion sites
and subsequent alteration of the chromatin dynamics have been
reported [7].
Subsequently, new live-cell imaging techniques were established that are based on the use of programmable DNA-binding
proteins, including zing finger protein (ZFP), transcription
activator-like effector (TALE), and, more recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPRassociated protein 9 (Cas9) (Fig. 1). The major advantage of programmable DNA-binding proteins is that target regions like RNA
or genomic DNA are recognized and marked in a sequence-specific
way.
ZFP fused with GFP under the control of ribosomal protein 5S
A promoter (RPS5Ap) was first used to label the 180 bp tandem
repeats of the centromere in the root meristems of A. thaliana
[8]. However, the ZFP system could not be used for imaging of
specific loci, such as 5S rDNA or HPT gene. Since the genomic and
chromatin content around the target region along with the
context-dependent interactions with neighboring zinc fingers
affects the DNA sequence recognition ability of ZFP for different
genomic targets [9, 10], the further application of ZFP for live-cell
imaging was substituted with TALEs. Similar to ZFPs, TALEs can
be programed to detect specific DNA sequences [11, 12]. Using
this feature, TALEs were successfully fused to the GFP to visualize
repetitive sequences in A. thaliana including telomeric sequences
and centromeric 180 bp repeats [13]. However, being a timeconsuming and laborious method due to reengineering of TALE
proteins for the targeting of each new genomic region, the live-cell
imaging techniques were improved to use a more user-friendly
method called CRISPR/Cas9.
The application of CRISPR/Cas9 for chromatin imaging was
first reported for the dynamic imaging of genomic loci in human
cell cultures [14]. The CRISPR/Cas9 consists of a Cas9 protein
and guide RNA (gRNA) scaffold. The gRNA is a fusion of crRNA
(CRISPR RNAs) and tracrRNA (trans-activating crRNA). Part of
crRNA which binds as a complementary strand to the foreign target
sequence is called protospacer. tracrRNA has a stem-loop structure
and supports the stability of Cas protein. The Cas9 protein contains
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Solmaz Khosravi et al.
GFP-fused chromatin proteins including histones, condensins,
and others [4]. Nonetheless, this method is unable to detect specific genomic loci. Later, fluorescent repressor/operator systems
were used to allow specific labeling of genomic regions in comparison to chromatin proteins [5]. In plants, this technique was applied
to A. thaliana to compare the ploidy level of guard cells and
elongated epidermal root cells [6]. However, this method is not
able to label predefined genomic regions as a random insertion of
the tandem operator repeats into the genome occurs after plant
transformation. In addition, methylation at operator insertion sites
and subsequent alteration of the chromatin dynamics have been
reported [7].
Subsequently, new live-cell imaging techniques were established that are based on the use of programmable DNA-binding
proteins, including zing finger protein (ZFP), transcription
activator-like effector (TALE), and, more recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPRassociated protein 9 (Cas9) (Fig. 1). The major advantage of programmable DNA-binding proteins is that target regions like RNA
or genomic DNA are recognized and marked in a sequence-specific
way.
ZFP fused with GFP under the control of ribosomal protein 5S
A promoter (RPS5Ap) was first used to label the 180 bp tandem
repeats of the centromere in the root meristems of A. thaliana
[8]. However, the ZFP system could not be used for imaging of
specific loci, such as 5S rDNA or HPT gene. Since the genomic and
chromatin content around the target region along with the
context-dependent interactions with neighboring zinc fingers
affects the DNA sequence recognition ability of ZFP for different
genomic targets [9, 10], the further application of ZFP for live-cell
imaging was substituted with TALEs. Similar to ZFPs, TALEs can
be programed to detect specific DNA sequences [11, 12]. Using
this feature, TALEs were successfully fused to the GFP to visualize
repetitive sequences in A. thaliana including telomeric sequences
and centromeric 180 bp repeats [13]. However, being a timeconsuming and laborious method due to reengineering of TALE
proteins for the targeting of each new genomic region, the live-cell
imaging techniques were improved to use a more user-friendly
method called CRISPR/Cas9.
The application of CRISPR/Cas9 for chromatin imaging was
first reported for the dynamic imaging of genomic loci in human
cell cultures [14]. The CRISPR/Cas9 consists of a Cas9 protein
and guide RNA (gRNA) scaffold. The gRNA is a fusion of crRNA
(CRISPR RNAs) and tracrRNA (trans-activating crRNA). Part of
crRNA which binds as a complementary strand to the foreign target
sequence is called protospacer. tracrRNA has a stem-loop structure
and supports the stability of Cas protein. The Cas9 protein contains
344
Solmaz Khosravi et al.