Toolkit Items

HY5 is an Arabidopsis thaliana basic leucine zipper (bZIP) transcription factor that directly binds light-responsive promoters and functions as a positive regulator of photomorphogenesis. It also mediates crosstalk between light signaling and the unfolded protein response (UPR) by negatively regulating UPR gene expression through promoter competition.

dCas9* is a Streptococcus pyogenes dCas9 variant carrying the PAM-binding mutation R1335K, engineered to eliminate PAM recognition and reduce toxicity in bacteria. In the cited study, dCas9* was also fused to the PhlF repressor to recover targetable transcriptional repression through a combined sgRNA target site and PhlF operator requirement.

SpCas9 is the Streptococcus pyogenes Cas9 CRISPR effector protein used for programmable genome editing and gene regulation. In the cited study, its activity was controlled indirectly by microRNA-dependent expression of the anti-CRISPR protein AcrIIA4, enabling cell-type-restricted activation of full-length Cas9, split-Cas9, and dCas9-VP64 variants.

dCas9*_PhlF is a bacterial CRISPR-based transcriptional switch comprising a non-toxic dCas9* variant with the R1335K PAM-binding mutation fused to the PhlF repressor. The fusion recovered DNA-binding-dependent repression and enabled sgRNA-programmed NOT gate behavior that depends on both an sgRNA target site and a PhlF operator.

CRYPTOCHROME-INTERACTING BASIC HELIX-LOOP-HELIX 1 (CIB1) is an Arabidopsis thaliana basic helix-loop-helix transcription factor that binds DNA and regulates transcriptional programs linked to cell elongation. It also physically interacts with the blue-light receptor CRYPTOCHROME 2 (CRY2) and has been described as promoting flowering.

eSpCas9 is an increased-fidelity Streptococcus pyogenes Cas9 nuclease variant used in the optimization of CRISPR-Cas9 cleavage specificity. In comparative analyses of high-fidelity SpCas9 enzymes, eSpCas9 served as one of the variants whose mutations were combined to generate hybrid HeFSpCas9 nucleases.

HeFSpCas9 denotes engineered Streptococcus pyogenes Cas9 high-fidelity nuclease variants that combine mutations from eSpCas9 and SpCas9-HF1. These variants were developed to optimize the balance between on-target cleavage activity and genome-editing specificity across different target sites.

SpCas9-HF1 is an engineered high-fidelity Streptococcus pyogenes Cas9 nuclease variant evaluated in comparative studies of increased-specificity SpCas9 enzymes. It is used for genome cleavage applications in which target-dependent optimization of specificity is required.

The basic helix-loop-helix (bHLH) domain is a highly conserved eukaryotic protein motif present in transcription factors. It contains a basic DNA-binding region and a helix-loop-helix region, supporting transcriptional regulatory functions across diverse physiological contexts.

Photoactivatable cyclic caged morpholino oligomers (ccMOs) are light-responsive antisense morpholino reagents engineered in a cyclic, caged format to suppress target binding until photoactivation. In the reported design, brief 405-nm illumination photocleaves the cage and restores antisense activity, enabling spatiotemporal regulation of gene expression.

PHYTOCHROME RAPIDLY REGULATED1 (PAR1) is a non-DNA-binding atypical helix-loop-helix (HLH) inhibitor implicated in light-regulated transcriptional control in plants. In the cited study, PAR1 suppresses cell elongation by interfering with the DNA-binding activity of the bHLH transcription factor CIB1.

Homology assisted CRISPR knock-in (HACK) is an engineering method used in Drosophila to convert existing GAL4 drivers into ShineGAL4 counterparts by replacing GAL4 with a GAL4 DNA-binding domain fused to a Magnet photoswitch. In the cited study, this generated light-responsive transcriptional drivers for optogenetic gene control.

AQTrip is an engineered EL222 variant carrying V41I, L52I, A79Q, and V121I substitutions in the blue-light-responsive LOV–HTH transcription factor. It stabilizes the photoactivated state and, in the reported study, oligomerizes without DNA and forms an EL222 dimer–DNA complex in the presence of DNA substrates.

The AUREO1 bZIP-LOV truncated construct (ZL) is an N-terminally truncated aureochrome-1 derivative that retains the bZIP DNA-binding region and the LOV photosensory domain. It binds DNA in a sequence-specific manner and undergoes a blue-light-induced conformational response measurable as an approximately 5% increase in hydrodynamic radius without a detectable change in secondary structure.

The full-length AUREO1 construct is a blue-light-responsive transcription factor from Vaucheria frigida that contains an N-terminal bZIP domain and a C-terminal LOV domain. In the reported study, the full-length protein binds DNA in a sequence-specific manner and undergoes an approximately 5% blue-light-induced increase in hydrodynamic radius.

Here, we report on the discovery of ligands for the DNA binding domain of MITF, using fragment-based screening (FBS) by nuclear magnetic resonance (NMR).

Here, we report the design of a protease-responsive conformationally inhibited system (PRCIS). By intramolecularly linking the free DNA-binding domains of ATF to confined dimerized regions, the transcriptional binding is conformationally inactivated. The function of DNA binding is reinstated upon proteolytic cleavage of linkages, activating the downstream gene expressions.