the orientation of loxP sequences (McLellan
et al. 2017). As proof of principle, Cre recombinase was divided into two parts that were fused
to CRY2 and CIB1, and an egfp reporter was
used to inform the recombination level in
response to blue light. A 15-min treatment
was enough to observe detectable levels of
fluorescence, which increased with longer
exposures to light (Kennedy et al. 2010). In
yeast, the light-dependent heterodimerization
between CRY2 and CIB1 has been also used to
control gene expression (Hughes et al. 2012).
Similarly, a chimeric transcription factor using
non-yeast domains such as LexA-DBD and
VP16-AD was designed (Fig. 12.2b), displaying
high levels of reporter gene expression in
response to blue light (Hughes et al. 2012).
Furthermore, CRY2/CIB1 photoactivated interaction was used to control sic1 gene expression,
which encodes for a regulator of the yeast
cell cycle whose degradation allows transition
between G1 and S phases (Verma et al. 1997;
Yang et al. 2013). Thus, yeast cells grew normally in darkness, but the optogenetic expression of a hyperstable Sic1p mutant led to the
accumulation of this protein and cell cycle
arrest, observing dramatic cell morphology
changes in response to 4 h of blue light (Hughes
et al. 2012). Recently, a similar approach was
developed in yeast using the CRY2/CIB1 interaction. In order to avoid the interference that
can exist by using endogenous building blocks,
the DBD of the Zif268 mammalian transcription factor was fused to the PHR domain of
CRY2 (An-Adirekkun et al. 2019). Furthermore,
a synthetic promoter was generated by placing
the 9-bp specific binding element of Zif68 in a
gal1 minimum promoter that lacks the GAL4binding upstream activating sequence (UAS),
which was previously demonstrated to work
orthogonally in yeast (McIsaac et al. 2013).
Characterization of the system included a variation of the number of Zif68-binding UAS element repetitions in the promoter as well as
different orientations, changes in light intensity, and several illumination treatments. Various reporter genes and expression levels of the
components were also tested, obtaining a wide
range of responses that even surpassed the
expression levels reached by native yeast promoters (An-Adirekkun et al. 2019). On the
other hand, a two-component optogenetic
expression system based on CRISPR/Cas9 technology was developed and implemented in
mammalian cells (Fig. 12.2d). To this end, a
catalytically inactive “dead” mutant of Cas9
(dCas9) was fused to CIB1 and different transactivation domains were fused to the CRY2PHR domain. In response to blue light, a transcription system was thus reconstituted activating gene expression depending on the guide
RNA (gRNA) targeting a specific promoter
(Fig. 12.2d) (Nihongaki et al. 2015a, b). Using
a luciferase reporter and three different gRNAs
that recognize a gal1 promoter, this system
showed high levels of bioluminescence in
response to illumination. This photoactivable
transcription system also allowed the spatial
expression of an mCherry fluorescent protein
(Nihongaki et al. 2015a, b). Importantly, synthetic expression systems based on CRISPR/
Cas9 allow easier activation of endogenous
genes because it is not necessary to modify the
promoters of the genes of interest. For this
reason, the authors probed their system to
induce transcription of several genes in mammalian cells, observing high transcript levels of
ascl1, myod1, nanog, and il1rn in response to
light using multiple gRNAs, showing the feasibility of simultaneously controlling the transcription of multiple genes (Nihongaki et al.
2015a, b). Considering the molecular size of
the Cas9 protein, the CRISPR/Cas9 photoactivatable transcription system was optimized. For
this, CIB1 was fused to the amino- and
carboxyl-terminus of the dCas9 protein, developing a new system called Light-Activated
CRISPR/Cas9 Effector (“LACE”). This approach
increased the expression of endogenous genes
in mammalian cell lines, with a precise spatiotemporal resolution (Polstein and Gersbach
2015). Besides its light-dependent heterodimerization with CIB1, CRY2 can oligomerize upon
illumination (Bugaj et al. 2013). Thereby, several protein clustering approaches have been
developed using this property, such as the
“CLICR” system which fused CRY2 to an adaptor that can bind specific proteins (Bugaj et al.
2015). In darkness, CRY2 monomers bind their
targets with low affinity; however, light-induced
302
V. Rojas et al.
et al. 2017). As proof of principle, Cre recombinase was divided into two parts that were fused
to CRY2 and CIB1, and an egfp reporter was
used to inform the recombination level in
response to blue light. A 15-min treatment
was enough to observe detectable levels of
fluorescence, which increased with longer
exposures to light (Kennedy et al. 2010). In
yeast, the light-dependent heterodimerization
between CRY2 and CIB1 has been also used to
control gene expression (Hughes et al. 2012).
Similarly, a chimeric transcription factor using
non-yeast domains such as LexA-DBD and
VP16-AD was designed (Fig. 12.2b), displaying
high levels of reporter gene expression in
response to blue light (Hughes et al. 2012).
Furthermore, CRY2/CIB1 photoactivated interaction was used to control sic1 gene expression,
which encodes for a regulator of the yeast
cell cycle whose degradation allows transition
between G1 and S phases (Verma et al. 1997;
Yang et al. 2013). Thus, yeast cells grew normally in darkness, but the optogenetic expression of a hyperstable Sic1p mutant led to the
accumulation of this protein and cell cycle
arrest, observing dramatic cell morphology
changes in response to 4 h of blue light (Hughes
et al. 2012). Recently, a similar approach was
developed in yeast using the CRY2/CIB1 interaction. In order to avoid the interference that
can exist by using endogenous building blocks,
the DBD of the Zif268 mammalian transcription factor was fused to the PHR domain of
CRY2 (An-Adirekkun et al. 2019). Furthermore,
a synthetic promoter was generated by placing
the 9-bp specific binding element of Zif68 in a
gal1 minimum promoter that lacks the GAL4binding upstream activating sequence (UAS),
which was previously demonstrated to work
orthogonally in yeast (McIsaac et al. 2013).
Characterization of the system included a variation of the number of Zif68-binding UAS element repetitions in the promoter as well as
different orientations, changes in light intensity, and several illumination treatments. Various reporter genes and expression levels of the
components were also tested, obtaining a wide
range of responses that even surpassed the
expression levels reached by native yeast promoters (An-Adirekkun et al. 2019). On the
other hand, a two-component optogenetic
expression system based on CRISPR/Cas9 technology was developed and implemented in
mammalian cells (Fig. 12.2d). To this end, a
catalytically inactive “dead” mutant of Cas9
(dCas9) was fused to CIB1 and different transactivation domains were fused to the CRY2PHR domain. In response to blue light, a transcription system was thus reconstituted activating gene expression depending on the guide
RNA (gRNA) targeting a specific promoter
(Fig. 12.2d) (Nihongaki et al. 2015a, b). Using
a luciferase reporter and three different gRNAs
that recognize a gal1 promoter, this system
showed high levels of bioluminescence in
response to illumination. This photoactivable
transcription system also allowed the spatial
expression of an mCherry fluorescent protein
(Nihongaki et al. 2015a, b). Importantly, synthetic expression systems based on CRISPR/
Cas9 allow easier activation of endogenous
genes because it is not necessary to modify the
promoters of the genes of interest. For this
reason, the authors probed their system to
induce transcription of several genes in mammalian cells, observing high transcript levels of
ascl1, myod1, nanog, and il1rn in response to
light using multiple gRNAs, showing the feasibility of simultaneously controlling the transcription of multiple genes (Nihongaki et al.
2015a, b). Considering the molecular size of
the Cas9 protein, the CRISPR/Cas9 photoactivatable transcription system was optimized. For
this, CIB1 was fused to the amino- and
carboxyl-terminus of the dCas9 protein, developing a new system called Light-Activated
CRISPR/Cas9 Effector (“LACE”). This approach
increased the expression of endogenous genes
in mammalian cell lines, with a precise spatiotemporal resolution (Polstein and Gersbach
2015). Besides its light-dependent heterodimerization with CIB1, CRY2 can oligomerize upon
illumination (Bugaj et al. 2013). Thereby, several protein clustering approaches have been
developed using this property, such as the
“CLICR” system which fused CRY2 to an adaptor that can bind specific proteins (Bugaj et al.
2015). In darkness, CRY2 monomers bind their
targets with low affinity; however, light-induced
302
V. Rojas et al.