Toolkit Items

15N and 1H liquid-state high-resolution NMR is an assay method used to detect light-induced photo-CIDNP signals in engineered Mr4511 flavoproteins. In the cited study, it reported nuclear hyperpolarization arising from a light-driven transient paramagnetic state in variants containing tryptophan at canonical or newly introduced positions.

AcrIIA4 is an anti-CRISPR protein characterized in Nicotiana benthamiana as an inhibitor of CRISPR/Cas9 function. In plant assays, it blocks Cas9-mediated site-directed mutagenesis and represses CRISPR/dCas-based transcriptional activation, including when delivered by Tobacco etch virus.

The Affymetrix ATH1 microarray is a transcript expression assay for genome-scale expression profiling in Arabidopsis thaliana. In the cited AtGenExpress study, it was used to generate a comprehensive transcript dataset across abiotic stress conditions including UV-B light, drought, and cold.

The all-optical framework for functional testing of opsin responsiveness in cardiac fibroblasts is an assay method described to evaluate whether virally introduced optogenetic actuators are functionally responsive in primary cFB. In the cited study, it provides an optical functional readout for opsin-expressing cardiac fibroblasts and is associated with co-culture conditions that can yield a light-sensitive excitable cardiac syncytium.

The allelic series of Cry mutants is a panel of 22 cryptochrome variants, each carrying a single amino acid substitution identified by random mutagenesis and a cell-based screen. The series was used to generate diverse deficiencies across CRY functions and to identify residues involved in protein-protein interaction and CRY2-specific repression.

AsLOV2-V416L is a point-mutated Avena sativa LOV2 photosensory domain variant with strongly decelerated dark-state recovery after photoactivation. In the reported screen, it exhibited a thermal reversion time constant of 4.3 × 10^3 s, corresponding to a 78-fold slower recovery than wild-type AsLOV2.

AsLOV2-V416T is a point-mutated Avena sativa LOV2 photosensory domain variant identified in a mutagenesis and fluorescence imaging-based screen for altered recovery kinetics. It exhibits markedly accelerated thermal reversion from the light-activated state, with a reported time constant of 2.6 s, 21-fold faster than wild-type AsLOV2.

The atomic force sensing technique is an assay method for dynamically probing protein conformational changes with microsecond time resolution. In the cited 1997 study, it was applied to light-induced conformational changes in bacteriorhodopsin.

Automated 96-well microplate illumination and measurement is an assay method for high-throughput optogenetic characterization of cultures under controlled light input. In the cited Saccharomyces cerevisiae workflow, it supported construction and characterization of split transcription factors containing cryptochrome and Enhanced Magnet light-sensitive dimerizers.

This Bayesian computational approach is a data-analysis method developed to improve prediction of split protein behavior by contextualizing errors inherent to experimental procedures. In the cited study, it was applied to pooled, sequencing-based screening data from split Cre recombinase constructs generated with optogenetic dimers, enabling comprehensive analysis of split sites across the protein.

The Bayesian optimization framework is a computational method built from high-throughput Lustro measurements to guide control of blue light-sensitive optogenetic systems. It uses data-driven learning, uncertainty quantification, and experimental design to identify light induction conditions for multiplexed regulation in Saccharomyces cerevisiae.

The Bessel beam stimulation source integrated into lattice light-sheet microscopy (LLSM) is an optical assay configuration that adds Bessel-beam-based optogenetic stimulation to LLSM without changing the microscope optical configuration. It enables three-dimensional photoactivation with improved spatiotemporal control and has been used to manipulate subcellular membrane ruffling and guide cell migration.

Bioreactor designs for optogenetic stimulation are light-delivery and culture-platform configurations used to stimulate optogenetic systems across experimental formats ranging from simple illumination set-ups to microscopy, microtiter plate, and bioreactor designs. These platforms support applications spanning single-cell stimulation to whole-culture illumination and can be integrated with automated measurement and stimulation for computer-controlled experiments.

This tool is a bioreactor-based assay platform coupled to automated cytometry measurements for monitoring intracellular protein levels and secretory stress during production of hard-to-secrete proteins. It was reported as a real-time measurement platform to identify an optimal secretory-stress regime during protein production.

Biosensing is mentioned only as an emerging assay-related strategy expected to shape future directions in the field. The supplied evidence does not define a specific biosensor modality, analyte, or experimental implementation.

Bisulfite pyrosequencing is an assay method used to quantify promoter CpG DNA methylation after bisulfite treatment. In the cited study, it was used to measure methylation in the CAMK1D, CRY2, and CALM2 promoter regions in peripheral blood from patients with type 2 diabetes and matched healthy controls.

Booster is a red-shifted genetically encoded FRET biosensor backbone generated by optimizing the order of fluorescent proteins and modulatory domains within a biosensor architecture. In the reported implementation, a Booster-PKA sensor enabled kinase activity readout in a spectral window compatible with CFP/YFP-based FRET biosensors and blue light-responsive optogenetic tools.

Caenorhabditis elegans light-induced coclustering (CeLINC) is a fluorescence-based optical binary protein-protein interaction assay for testing whether two proteins interact in vivo in C. elegans. It uses light-induced coclustering as the assay readout for protein association.

Cas12aVIP is a rapid visual nucleic acid detection assay that integrates recombinase polymerase amplification, a CRISPR/Cas12a detection system, and a PMNT plus single-stranded DNA color reporter. It was proposed for visual readout of target nucleic acids in a format intended to be rapid and directly observable.

A cDNA microarray is a gene-expression profiling assay used in Arabidopsis to examine how phyA pathway mutations affect far-red light control of genome-wide expression. In the cited study, it was applied to profile transcriptional responses associated with phytochrome A signaling and to compare mutant expression patterns.

Cell-free chromatin immunoprecipitation (cfChIP) is an assay method that immunoprecipitates histone mark-associated cell-free chromatin from blood plasma. In the cited study, cfChIP targeting H3K36me3-associated cfDNA was used with droplet digital PCR to infer transcriptional activity of specific genes, including EGFR, in the cells that released the cfDNA.

cfDNA fragmentomics evaluation is an assay method that analyzes plasma cell-free DNA fragment length distributions and fragment end motifs to identify signatures associated with active gene expression. In a 2024 study, integrating short-fragment frequency with end-motif information improved enrichment for highly expressed genes in plasma samples from lung cancer patients and healthy individuals.

Chlorophyll fluorescence lifetime measurements are an optical assay method used to detect free chlorophyll. In Arabidopsis chaos leaves, this method detected free chlorophyll before visible symptoms of oxidative stress appeared.

Chromatin immunoprecipitation sequencing (ChIP-seq) is an assay method that combines chromatin immunoprecipitation with sequencing-based genomic localization to map protein-associated genomic regions. In the cited study, it was used to identify genome-wide ZFHX3-binding sites in suprachiasmatic nucleus chromatin, revealing occupancy concentrated near transcription start sites and co-localization with known histone modifications.

Chromatin in vivo imaging is identified in a 2018 review as a CRISPR/Cas9-based epigenetic technique for imaging chromatin in living systems. The supplied evidence supports its existence as a method category within the CRISPR/Cas9 epigenetics toolkit, but does not describe a specific construct, protocol, or performance profile.

The CIb1 promoter is a Bombyx mori gene promoter characterized by firefly luciferase reporter assays in the ovary-derived BmN cell line. Deletion analysis identified promoter fragments with transcriptional activity, including an 82 bp region from -72 to +10 nt that retains basic promoter function, and an upstream segment with strong negative regulatory effects.

CIDNP (chemically induced dynamic nuclear polarization) is a phenomenon in which chemical reactions generate nuclear hyperpolarization. Photo-CIDNP is the light-driven form of this phenomenon and is discussed in electron-transfer systems that often use flavins as electron acceptors.

cLIPS2 is a light-responsive multi-component switch identified from small libraries of cLIPS1 variants in a higher-throughput yeast screen. It binds human eIF4E in a light-dependent manner in vitro and inhibits translation in vivo in yeast harboring human eIF4E, with improved optical control relative to screened cLIPS1 variants.

Closed-loop all-optical neuromodulation is a multi-component optogenetic strategy that combines optical actuators with optical indicators. It is proposed to enable feedback-controlled neuromodulation using light for both perturbation and activity readout.

Comprehensive insertion libraries are a high-throughput engineering method in which many insertion variants are generated and screened. In the cited context, they are discussed as an approach that could accelerate creation of stimulus-responsive receptor–protein chimeras.

Computational protein design is an engineering methodology described in a 2018 review as a next-generation tool for expanding synthetic biology applications. The supplied evidence frames it as a design approach used alongside phage display and high-throughput binding assays rather than as a single molecular reagent.

Confocal microscopy is an in vivo fluorescence imaging assay method described as part of microscopy platforms tailored to larval zebrafish research. In the cited review context, it is used with fluorescent probes for real-time monitoring of cell identity, fate, and physiology in living larvae, including pancreatic and islet studies.

Cryogenic-temperature ENDOR spectroscopy is a spectroscopic assay method applied to LOV domains to interrogate the local environment of the flavin mononucleotide (FMN) cofactor. It does so by measuring temperature-dependent hyperfine couplings associated with hindered rotation of the methyl group attached at C(8) of the FMN isoalloxazine ring.

The custom Python-based API is a software interface for assembling automation workflows on an open-source microplate reader. It enables programmable control of automated assay protocols for an instrument demonstrated for full-spectrum absorbance, fluorescence emission detection, and in situ optogenetic stimulation.

dcFCCS is a dual-color fluorescence cross-correlation spectroscopy assay method used to quantify interactions relevant to cGAS phase separation. In the cited study, it was applied to systematically examine binding among cGAS, double-stranded DNA, and accessory proteins in relation to condensate formation and enzymatic activity.

The directional reaching for water behavioral framework is a head-fixed mouse assay in which animals make center-out-like reaches toward water droplets presented at multiple spatial locations. It enables structured study of cortex-dependent reaching, sensorimotor processing, and decision making, and has been used with optogenetic motor cortex inactivation.

The droplet microfluidic platform is an assay method for screening and separating cell populations based on the in vivo fluorescence response of expressed biosensors after addition of an exogenous analyte. It was applied to HeLa-cell genetic linker libraries for genetically encoded Zn2+ sensors to assess library diversity and detect response heterogeneity.

The dual transient visible absorption (visTA)/FSRS set-up is a broadband time-resolved spectroscopic assay that combines transient visible absorption with femtosecond stimulated Raman spectroscopy. It covers approximately 200–2200 cm^-1 and supports pump–probe delays from a few femtoseconds to several hundreds of microseconds after actinic light excitation, enabling monitoring of photoinduced dynamics.

EGxxFP is a Cas9 reporter construct used in split-Cas9 synthetic circuits to convert successful Cas9 reconstitution into a fluorescent readout. In the cited 2023 Scientific Reports study, it reported cellular states and events including cancer epithelial origin, epithelial-to-mesenchymal transition, and cell-cell fusion.

Electron paramagnetic resonance (EPR) is an assay method used to monitor light-induced paramagnetic signals. In the cited study, it measures the kinetics of appearance and disappearance of signals assigned to P700+ and iron-sulfur protein(s) at low temperature.

Electron-electron double resonance (ELDOR) spectroscopy is a structural assay method that, when combined with site-directed spin labelling, was used to chart light-induced structural transitions in the engineered LOV histidine kinase YF1. In the cited study, it provided pairwise distance information used to model blue-light-driven quaternary rearrangements in a signaling photoreceptor.

Electron-transfer/higher-energy collision dissociation (EThcD) is a top-down mass spectrometry fragmentation method used in a combined workflow with 213 nm ultraviolet photodissociation (UVPD) to characterize covalent insulin dimers. In the cited study, this workflow identified cross-link chemical composition and, with MS3 analysis of informative MS2 fragments, enabled residue-level localization of interchain cross-link sites.

Electrophysiology is used as a functional assay in a multimodal study of gasdermin D pore behavior, alongside optogenetic tools and live-cell fluorescence biosensing. In the cited work, it supports measurement of pore conductance dynamics and the conclusion that gasdermin pores show phosphoinositide-dependent, repeated fast opening-closing behavior.

The endometrial receptivity assay (ERA) is a clinical assay intended to assess endometrial receptivity during the window of implantation. The supplied evidence indicates that, as a current test of the window of implantation, it does not consistently improve clinical outcomes measured by live birth rates.

Endometrial thickness measurement is a current clinical assay method used as a marker of the window of implantation and endometrial receptivity. The cited evidence indicates that, as a current test of the window of implantation, it does not consistently improve clinical outcomes measured by live birth rates.

EndoV-seq is an assay method reported for genome-wide profiling of adenine base editor specificity. The available evidence supports its use as a functional assay to identify where adenine base editing occurs across the genome.

Epigenetic element screening is described in a 2018 review as a CRISPR/Cas9-based epigenetic technique. The supplied evidence establishes only that it belongs to the set of CRISPR/Cas9-enabled approaches used in epigenetics, without further methodological detail.

This assay-method profile describes genomic analysis used to study light-regulated gene expression in plants. The cited review states that such studies revealed epigenetic regulation, massive transcriptome reprogramming in response to light, and genome-wide binding sites for pivotal light-signaling transcription factors.

An expressed sequence tag-based microarray is a microarray expression-profiling assay used to measure genome-scale transcript patterns in Arabidopsis. In the cited study, it was applied to profile gene expression underlying light-controlled development across different light conditions.

Field-domain rapid-scan EPR at 240 GHz is a high-frequency electron paramagnetic resonance assay method reported for studying protein functional dynamics at room temperature. The available evidence identifies it specifically as a field-domain rapid-scan EPR approach operating at 240 GHz.