Toolkit Items

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.

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.

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.

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.

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.

This entry describes delivery-system choice as a genome-editing optimization strategy rather than a single defined molecular reagent. In the cited 2022 review, delivery-system selection is presented as one approach for improving the efficiency of CRISPR/Cas-induced mutations.

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.

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.

Fluorescence recovery after photobleaching (FRAP) is proposed as a functional assay readout for liquid-like molecular mobility within the pathological condensate termed the addivosome. In this context, FRAP is intended to detect restoration of mobility, or reliquefaction, during compound screening.

Fluorescent polarization is an assay method provided as a protocol for validating, improving, and using newly designed photoswitches in the context of LOV2-based optogenetic engineering. In the cited source, it is presented alongside phage display and microscopy as part of the experimental toolkit for photoswitch development.

FRASE, also described as FRASE-bot, is a computational fragment-based ligand discovery method that mines 3D ligand–protein complex structures to build a database of fragments in structural environments. It screens this database against a target protein, seeds the target structure with relevant ligand fragments, and uses a neural network to prioritize fragments with the highest likelihood of being native binders.

FUN-LOVSP-Hph is a blue-light-responsive yeast optogenetic switch variant of FUN-LOVSP that includes a hygromycin resistance cassette for selection after genome integration. It drives light-induced reporter expression and was reported to outperform the original FUN-LOV system in BY4741 yeast while remaining functional in a wine yeast strain.

Genetic screens in Arabidopsis thaliana are a plant genetics method used to identify components of light-responsive signal transduction pathways. The cited evidence states that several laboratories devised such screens to dissect pathways associated with various photoreceptor systems.

H3K36me3 cell-free chromatin immunoprecipitation sequencing (cfChIP-seq) is a plasma-based assay that establishes a personal gene expression profile from cell-free chromatin. In the cited study context, it functions as a reference enrichment assay for active genes in liquid biopsy samples.

H3K36me3 cfChIP followed by droplet digital PCR is a cell-free chromatin immunoprecipitation assay that enriches plasma cfDNA associated with the transcription-linked histone mark H3K36me3 and then quantifies specific alleles by ddPCR. In a 2021 NSCLC study, it detected greater enrichment of EGFR-L858R fragments than EGFR wild-type fragments, providing proof of principle for identifying tumor-specific transcriptional activity of mutated alleles.

High-throughput screening is an assay method cited in microbial biotechnology literature as part of the CRISPR/Cas toolbox for evaluating variants generated by multiplexed engineering. In the supplied evidence, it is presented as a screening approach associated with CRISPR/Cas-based metabolic engineering and with development of new dynamic systems.

The high-throughput online monitoring system with an LED array is an assay platform for screening light-controlled gene expression conditions by individually illuminating each well in a multiwell format. In the cited yeast study, it was used with photocaged Cu2+ to regulate the Cu2+-inducible pCUP1 promoter from Saccharomyces cerevisiae and monitor eYFP expression.

The hypocotyl elongation screen for ethylene-insensitive Arabidopsis mutants is a phenotypic assay based on ethylene-mediated inhibition of hypocotyl elongation in dark-grown seedlings. Arabidopsis mutants that remain tall despite treatment with high concentrations of ethylene are identified as ethylene-insensitive.

LC-MS analysis of fittest binders is an assay method used with small combinatorial libraries of self-assembled proteomimetics (SAPs) to identify enriched target binders after affinity selection by liquid chromatography–mass spectrometry. In the cited SAP study, this workflow was applied in the context of target-directed selection from self-assembled PNA-peptide conjugate libraries.

The light-inducible split Cre recombinase is an optogenetic multi-component switch in which split Cre recombinase fragments are coupled to light-inducible dimerization modules to achieve inducible post-translational control of Cre activity. It was characterized by comprehensive screening of split sites across the Cre protein using a pooled, sequencing-based domain insertion profiling approach.

Light-switchable transcription factors are multi-component optogenetic regulators reported in a 2023 Nature Communications study. The available evidence indicates that they were obtained by direct screening in mammalian cells and are designed to enable light-dependent control of transcription factor activity.

Microscopy is a protocolized assay method included alongside fluorescent polarization and phage display in a 2020 methods source on engineering and applying LOV2-based photoswitches. In that context, it is used as part of the experimental workflow for validating, improving, and using light-responsive optogenetic switches built on the LOV2 domain.

Multiplexed engineering refers here to the use of the CRISPR/Cas toolbox for simultaneous genome engineering tasks in microbial biotechnology. The cited review places this approach in the context of metabolic engineering and high-throughput screening for production of chemicals and natural compounds.

The NanoLuc luciferase reporter assay is a bioluminescent functional reporter assay used to test guide RNA efficiency. In the cited study, it served as a pre-screening step to identify optimal guide RNAs before downstream cell-based experiments with an optogenetic CRISPR-dCas9 LITE system.

NCBI sequence screening for 2A/2A-like occurrence is a computational survey method that updates the distribution of viral 2A and 2A-like sequences by screening sequences deposited in the National Center for Biotechnology Information database. In the cited 2021 review, this approach identified 69 newly reported 2A-like occurrences across multiple virus groups.

The nMag/pMag photodimerization system, also called Magnets photosensors, is a light-controlled protein-domain pair that mediates heterodimerization. Reported engineering work altered its light sensitivity and tuned its light-activity dose-response behavior through directed evolution and high-throughput screening.

The open-source microplate reader is a low-cost, open-source assay platform for automated plate-based measurements. It was designed, constructed, validated, and benchmarked to support full-spectrum absorbance detection, fluorescence emission detection, in situ optogenetic stimulation, and stand-alone touch screen programming of assay protocols.

Orthogonal degrons are bacterial construct patterns used in tunable degradation systems to direct targeted proteolysis of proteins of interest. The cited literature places them among recent advances that enable large screens and functional interrogation through regulated protein degradation.

Phage display is an assay and selection method used during engineering workflows for light-responsive protein tools. In the cited context, it is applied alongside computational protein design and high-throughput binding assays in development of LOV2-based optogenetic systems such as improved light-induced dimers.

PHPLCδ1 is a phosphoinositide-binding pleckstrin homology domain used as a membrane-associated fluorescence probe. In the cited 2019 C. elegans zygote study, its reported PIP2 signal was interpreted as reflecting general plasma membrane localization rather than selective enrichment in a distinct PIP2 microdomain.

The pooled library approach is an engineering method for rapid generation and parallel screening of nearly all possible split-protein constructs, with sequencing-based readout. In the cited application, it was used with optogenetic dimers to comprehensively map split-site behavior across Cre recombinase and support inducible post-translational control design.

Screening based on selective labeling is identified in a 2023 review as an available genetic engineering tool within Aspergillus genome engineering workflows. The supplied evidence supports only that it is used as a screening approach associated with Aspergillus genetic technology, without operational or performance details.

SIBR is an inducible RNA-based regulatory element proposed to provide universal control of gene expression and gene regulation in bacteria. In the reported SIBR-Cas configuration, it regulates CRISPR-Cas counterselection with a temporal delay that improves genome editing in non-model bacterial hosts.

SIBR-Cas, termed Self-splicing Intron-Based Riboswitch-Cas, is a multi-component bacterial CRISPR genome engineering system that provides inducible control over CRISPR-Cas counterselection. It is reported to delay counterselection to permit editing events and has been applied to gene knockout in bacteria with poor homologous recombination systems.

Touchscreen-equipped operant conditioning chambers are a behavioral assay platform for measuring visual pairwise discrimination and reversal learning. In the cited 2023 study, they were used to quantify cognitive performance in Sprague Dawley rat offspring after gestational exposure to high-THC cannabis smoke.

Whole-genome screening of gene knockout mutants in Toxoplasma gondii is a CRISPR/Cas9-based assay method for generating and interrogating genome-scale loss-of-function mutant populations. The cited review identifies this as a recent use of CRISPR/Cas9 in an apicomplexan parasite.