all, the expression levels were increased and the
background activity reduced by optimizing the
promoter region including changes of the number of UAS, the length of the spacer between
these sequences, and the core promoter. In
addition, the concentration ratio between the
GAVPO transcription factor and the luciferase
reporter was also optimized (Ma et al. 2013).
Based on VVD blue light-dependent homodimerization, an optimized system was developed receiving the name “Magnets.” This is an
optogenetic system where two Neurospora
VVD molecules are fused to complementary
protein domains, avoiding the generation of
non-functional homodimers. To achieve this,
point mutations were introduced in the interface of the VVD LOV domain that is exposed to
the solvent. Specifically, neutral residues such
as isoleucine 52 (Ile52) and methionine 55
(Met55) were exchanged by charged amino
acids, generating a positive component
(pMag) and a negative component (nMag).
Thus, the formation of functional heterodimers
is optimized in response to blue light, minimizing homodimers by electrostatic repulsion.
“Magnets” were evaluated by light-induced
reconstitution of a split luciferase protein,
showing high bioluminescence levels with the
pMag/nMag pair (Kawano et al. 2015). Afterward, the same group reported the use of the
“Magnets” approach to Cre recombinase reconstitution by blue light treatment in mammalian
cells (Kawano et al. 2016). Using a luc reporter
as outcome of the Cre-mediated recombination, the photoactivatable Cre was extensively
verified using different loxP variants, cell lines,
light intensities, and light exposures (Kawano
et al. 2016). Interestingly, this optogenetic system showed better induction levels than a similar system based on CRY2/CIB1 interaction
(Kawano et al. 2016). Overall, high levels of
DNA recombination were obtained by this system, even showing activation in mice livers
(Kawano et al. 2016). In the same context,
“Magnets” dimerization domains were used to
control the reconstitution of a split Cas9 protein in mammalian cells (Nihongaki et al.
2015a, b). In this way, a specific editing of
genetic material was light-induced by activation of endogenous repair pathways. The activity of this optogenetic CRISPR/Cas9 system is
rapidly turned off in darkness and showed spatial activation defined by certain illumination
patterns (Nihongaki et al. 2015a, b). Likewise,
the “Magnets” approach was also utilized to
develop a light-inducible transcription system
through the reassembly of an orthogonal RNA
polymerase (RNAP) in E. coli. To this end, the
RNAP from the T7 virus was divided and each
part fused to pMag and nMag, generating different variants of this optogenetic system and
showing high induction of a mCherry reporter
(Baumschlager et al. 2017).
Interestingly, VVD photocycle mutants
could be also applied in optogenetics. By
mutating specific VVD residues, different variants of the protein that display altered photocycle lengths have been generated (Dasgupta
et al. 2015). For instance, the mutagenesis of
Ile74 and Ile85 by valine (Val) residues led to a
version with faster reversion between activated
and inactivated states, impairing the biological
function of VVD as shown by the incapacity of
the strain to attenuate gene expression and to
respond to increasing light intensities (Dasgupta et al. 2015). On the other hand, VVD
was also modified (Met135Ile/Met165Ile) to
generate a protein in which the stability of the
photo-adduct was 10 times longer (Dasgupta
et al. 2015). In that context, one of the main
advantages of optogenetic tools is the high temporal resolution that can be reached, permitting
the assessment of transient biological phenomena. For this reason, the light-inducible systems
reported so far have preferred photoreceptors
with fast-cycling kinetics, discarding the massive use of proteins such as FKF1. Nevertheless,
the development of slow-cycling versions could
be useful to induce cellular processes that are
needed to be permanent overtime with only a
brief illumination input.
Despite its capacity of light-induced homodimerization, VVD can also heterodimerize
with the transcription factor White Collar 1
(WC-1). In N. crassa, WC-1 interacts with the
WC-2 transcription factor by direct proteinprotein interaction via a different type of PAS
domain, forming the White-Collar Complex
(WCC) that commands circadian gene expression (Linden and Macino 1997). Additionally,
12 Exploiting Fungal Photobiology as a Source of Novel Bio-blocks for Optogenetic Systems
307
background activity reduced by optimizing the
promoter region including changes of the number of UAS, the length of the spacer between
these sequences, and the core promoter. In
addition, the concentration ratio between the
GAVPO transcription factor and the luciferase
reporter was also optimized (Ma et al. 2013).
Based on VVD blue light-dependent homodimerization, an optimized system was developed receiving the name “Magnets.” This is an
optogenetic system where two Neurospora
VVD molecules are fused to complementary
protein domains, avoiding the generation of
non-functional homodimers. To achieve this,
point mutations were introduced in the interface of the VVD LOV domain that is exposed to
the solvent. Specifically, neutral residues such
as isoleucine 52 (Ile52) and methionine 55
(Met55) were exchanged by charged amino
acids, generating a positive component
(pMag) and a negative component (nMag).
Thus, the formation of functional heterodimers
is optimized in response to blue light, minimizing homodimers by electrostatic repulsion.
“Magnets” were evaluated by light-induced
reconstitution of a split luciferase protein,
showing high bioluminescence levels with the
pMag/nMag pair (Kawano et al. 2015). Afterward, the same group reported the use of the
“Magnets” approach to Cre recombinase reconstitution by blue light treatment in mammalian
cells (Kawano et al. 2016). Using a luc reporter
as outcome of the Cre-mediated recombination, the photoactivatable Cre was extensively
verified using different loxP variants, cell lines,
light intensities, and light exposures (Kawano
et al. 2016). Interestingly, this optogenetic system showed better induction levels than a similar system based on CRY2/CIB1 interaction
(Kawano et al. 2016). Overall, high levels of
DNA recombination were obtained by this system, even showing activation in mice livers
(Kawano et al. 2016). In the same context,
“Magnets” dimerization domains were used to
control the reconstitution of a split Cas9 protein in mammalian cells (Nihongaki et al.
2015a, b). In this way, a specific editing of
genetic material was light-induced by activation of endogenous repair pathways. The activity of this optogenetic CRISPR/Cas9 system is
rapidly turned off in darkness and showed spatial activation defined by certain illumination
patterns (Nihongaki et al. 2015a, b). Likewise,
the “Magnets” approach was also utilized to
develop a light-inducible transcription system
through the reassembly of an orthogonal RNA
polymerase (RNAP) in E. coli. To this end, the
RNAP from the T7 virus was divided and each
part fused to pMag and nMag, generating different variants of this optogenetic system and
showing high induction of a mCherry reporter
(Baumschlager et al. 2017).
Interestingly, VVD photocycle mutants
could be also applied in optogenetics. By
mutating specific VVD residues, different variants of the protein that display altered photocycle lengths have been generated (Dasgupta
et al. 2015). For instance, the mutagenesis of
Ile74 and Ile85 by valine (Val) residues led to a
version with faster reversion between activated
and inactivated states, impairing the biological
function of VVD as shown by the incapacity of
the strain to attenuate gene expression and to
respond to increasing light intensities (Dasgupta et al. 2015). On the other hand, VVD
was also modified (Met135Ile/Met165Ile) to
generate a protein in which the stability of the
photo-adduct was 10 times longer (Dasgupta
et al. 2015). In that context, one of the main
advantages of optogenetic tools is the high temporal resolution that can be reached, permitting
the assessment of transient biological phenomena. For this reason, the light-inducible systems
reported so far have preferred photoreceptors
with fast-cycling kinetics, discarding the massive use of proteins such as FKF1. Nevertheless,
the development of slow-cycling versions could
be useful to induce cellular processes that are
needed to be permanent overtime with only a
brief illumination input.
Despite its capacity of light-induced homodimerization, VVD can also heterodimerize
with the transcription factor White Collar 1
(WC-1). In N. crassa, WC-1 interacts with the
WC-2 transcription factor by direct proteinprotein interaction via a different type of PAS
domain, forming the White-Collar Complex
(WCC) that commands circadian gene expression (Linden and Macino 1997). Additionally,
12 Exploiting Fungal Photobiology as a Source of Novel Bio-blocks for Optogenetic Systems
307