Toolkit Items

AsLOV2 is the light-oxygen-voltage 2 photosensory domain from Avena sativa phototropin 1 used as a blue-light-responsive actuator in engineered fusion proteins. Blue-light activation drives allosteric conformational extension involving sequential unfolding of the N-terminal A'α helix and the C-terminal Jα helix, enabling conformational uncaging and related optogenetic control.

The CRISPR/Cas9 system is a multi-component genome engineering platform derived from a bacterial defense system that uses Cas9 and guide RNA to manipulate genomic loci in living cells. It has been widely adopted for mutagenesis and genome research, with reported applications spanning basic biology, biotechnology, agriculture, medicine, epigenetic perturbation, and disease models.

Prime editing is mentioned in the cited review as part of the broader set of genome-editing approaches considered in bacterial genome engineering. The supplied evidence does not describe its molecular architecture, target scope, or editing outcomes.

Chimeric antigen receptor T-cell (CAR-T) therapy is a novel form of adoptive cellular immunotherapy that involves modifying autologous T cells to recognize and target tumor-associated antigens (TAAs) on malignant cells, independent of major histocompatibility complex (MHC) restriction.

Transcription activator-like effector nucleases (TALENs) are programmable site-specific nucleases used for genome editing. The supplied evidence describes them as artificial systems with customizable DNA-binding motifs that can be designed to target specific genomic loci, bind practically anywhere in the genome, and cleave double-stranded DNA.

Zinc finger nucleases (ZFNs) are programmable site-specific nucleases that use customizable DNA-binding motifs to target specific genomic loci for genome manipulation. The supplied evidence also places ZFNs among molecular tools used to alter gene expression and documents their use for gene knockout in sea urchins.

Chemogenetics is an engineering method in which target proteins are genetically engineered to interact with a designed chemical partner with high selectivity. It is used as a chemical-input strategy to manipulate protein or receptor function in cells and has also been used alongside optogenetics to perturb cellular structures such as specific microtubule subtypes.

Emerging synthetic biology tools, such as CRISPR-based transcriptional control, high-throughput screening, and machine learning-assisted promoter design, are enabling the creation of tunable, orthogonal promoters suited for complex multigene expression.

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.

CRY2-BIC1 is a multi-component blue-light-dependent interacting protein pair derived from Arabidopsis thaliana cryptochrome 2 (CRY2) and Blue-light Inhibitor of Cryptochromes 1 (BIC1). It functions as a light-input protein interaction switch, and phage-assisted continuous evolution was applied to increase the dynamic range of the blue-light-dependent CRY2-BIC1 interaction.

mOptoT7 is a mammalian optogenetic transcription system composed of a split T7 RNA polymerase fused to the blue-light-inducible nMag/pMag Magnets photodimerization system. Blue light drives reconstitution of the split polymerase to activate transcription from orthogonal T7 promoters in mammalian cells, and the system has been used to produce protein-coding mRNA, shRNA, and the Pepper RNA aptamer.

Acoustic reporter genes (ARGs) have enabled imaging of gene expression with ultrasound.

The CpbHLH gene family is the set of basic helix-loop-helix transcription factor genes identified in Chimonanthus praecox. A genome-wide study reported 131 CpbHLH genes distributed across 11 chromosomes and characterized their expression across tissues and flower developmental stages.

Directed evolution is an engineering method that improves biological tool performance by iteratively selecting functional protein variants. In the cited split fluorescent protein study, it was demonstrated as one of two approaches used to improve split fluorescent proteins, contributing to brighter split sfCherry3 variants.

the potential of lipid nanoparticle (LNP)-based messenger RNA (mRNA) vaccines to revolutionize HIV prevention

Innovations in AAV engineering, such as capsid modification (chemical conjugation, rational design, directed evolution), self-complementary genomes, and tissue-specific promoters (e.g., MHCK7), enhance muscle tropism while mitigating immunogenicity and off-target effects.

The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years.

The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

Neuroligin-1 sfCherry3 Linker Across Synaptic Partners (NLG-1 CLASP) is a red trans-synaptic marker built from split sfCherry3 and neuroligin-1-based linkage across synaptic partners. It was reported to enable multiplexed visualization of neuronal synapses in living Caenorhabditis elegans.

This directed protein evolution generates several gain-of-function OsTIR1 variants, including S210A, that significantly enhance the overall degron efficiency.

PACE (Phage Assisted Continuous Evolution) is an engineering method used in this study to evolve cryptochrome properties. In the cited work, it was applied to increase the dynamic range of the blue-light-dependent interaction between Arabidopsis thaliana CRY2 and BIC1.

The PAS domain superfamily comprises widespread sensory/regulatory protein domains present in archaea, bacteria, and eukaryota. Available evidence characterizes PAS domains through combined sequence, structure, and dynamics analysis, showing that functional groups can be differentiated by conserved-residue fluctuation patterns and statistically distinct vibrational behaviors.

RGEPO1, targeted to the extracellular membrane, and RGEPO2, localized in the cytoplasm, exhibited positive K+-specific fluorescence response with affinities of 2.4 and 43.3 mM in HEK293FT cells, respectively.

RGEPO1, targeted to the extracellular membrane, and RGEPO2, localized in the cytoplasm, exhibited positive K+-specific fluorescence response with affinities of 2.4 and 43.3 mM in HEK293FT cells, respectively.

In this study, we developed two novel red genetically encoded potassium indicators (RGEPOs), RGEPO1 and RGEPO2.

Innovations in AAV engineering, such as capsid modification (chemical conjugation, rational design, directed evolution), self-complementary genomes, and tissue-specific promoters (e.g., MHCK7), enhance muscle tropism while mitigating immunogenicity and off-target effects.

Single-headed kinesin molecular motors with optically enhanced clustering are engineered motors for microtubule-based active fluids that allow light-dependent control of extensile active stress. In the reported system, they support precise and repeatable spatiotemporal patterning of activity and rapid, reversible switching between flowing and quiescent states.

Split sfCherry3 variants are engineered split red fluorescent proteins derived from sfCherry through directed evolution. They were reported to show substantially enhanced overall brightness and to support visualization of endogenous proteins and neuronal synapses.

Here we use rational protein design and directed evolution to develop two new ARGs that can be distinguished from each other based on their acoustic pressure-response profiles, enabling 'two-tone' ultrasound imaging of gene expression.

sophisticated logic-gated circuits

Channelrhodopsins are light-activated ion channels from algae used as optogenetic tools to control membrane potential. Reported channelrhodopsin variants conduct either cations or anions, enabling light-driven depolarization or hyperpolarization.

Optogenetic networks are multi-component light-controlled genetic devices built from genetically encoded photosensitive proteins to regulate cellular behaviors and biological processes. They are developed to provide user-defined, minimally invasive, and spatiotemporally precise control for biomedical applications, including human disease therapy.

CRISPR-Cas-mediated genome editing is a programmable genome-editing approach discussed here in the context of bacterial systems. The cited review summarizes the main approaches for bacterial CRISPR-Cas editing and the difficulties associated with applying these systems in bacteria.

Domain fusion is a protein engineering method in which protein domains are fused or split to improve existing protein functions or create novel functions. In the supplied evidence, it is described as a general strategy for expanding CRISPR-Cas9 applications.

Gene editing technology is an engineering method used for animal model construction. The supplied evidence specifically states that it has been widely applied to nonhuman primate model generation in recent years.

The gene-pyramiding approach is an insect resistance management strategy used in the U.S. to combat the evolution of insect resistance to Bt crops. The supplied evidence identifies it as one of the two main IRM strategies alongside the high dose/refuge approach, but does not provide further mechanistic detail.

Protein splitting is a protein engineering method in which proteins are modified through domain fusion or splitting to improve existing functions or develop novel functions. In the provided evidence, it is discussed as a strategy relevant to expanding CRISPR-Cas9 applications.

Rational design is a protein engineering method used to improve or create novel protein functions. In the supplied evidence, it is discussed as one of the strategies used to expand CRISPR-Cas9 applications.

Recently, an evolutionarily conserved class of ribosomal oxygenases (ROX) that catalyze the hydroxylation of specific residues in the ribosome has been identified in bacteria.

Standardisation is an engineering method in synthetic biology in which engineering principles are applied to genetic manipulation workflows. The cited literature states that standardisation, together with key technical advances, enabled major gains in the speed and accuracy of genetic manipulation.

Vip3Aa is a Bacillus thuringiensis (Bt) insecticidal protein discussed in the context of Bt corn and cotton targeting Spodoptera frugiperda. The supplied evidence establishes its relevance as one of the Bt proteins used against this pest, but does not describe domain architecture, molecular mechanism, or engineered variants.

Countermeasures now integrate ... Domain III chimerization (e.g., Cry1A.105)

CRISPR/Cas is a DNA manipulation and genome editing technology derived from a prokaryotic immune system. It recognizes and destroys invading genetic elements and has progressed from bench research toward clinical practice.

Cyanobacteriochromes are cyanobacterial photoreceptor proteins built around a bilin-binding GAF domain related to phytochromes. They sense colors of light distinct from canonical phytochromes and have been applied in synthetic biology, including as the basis for an engineered near-infrared fluorescent protein tag.

Cyanobacteriochromes are photoswitchable protein domains from cyanobacteria that sense light across a broad spectral range from the UV to the near infra-red. In the cited 2022 work, they were engineered into light-inducible dimer pairs that support orthogonal control with red, green, and blue light.

DspA, also called Hik33, is a sensor histidine kinase from Synechocystis sp. strain PCC6803. It functions in signal transduction that controls photosynthetic and high-light-responsive gene sets and contributes to adaptation of cellular metabolism and growth to environmental light conditions.

MAPK signaling cascades are reported in zebrafish cells as light-activated signaling pathways that regulate gene expression. In the cited study, this light-responsive MAPK activity is linked to expression of z64Phr and zCry1a and to shared regulation of DNA repair and circadian clock entrainment programs.

Microbial rhodopsins are rhodopsin-based optogenetic protein domains that are activated by visible light. The supplied evidence identifies them as the most easily and most widely applied optogenetic tools in C. elegans.

The oat phy-CAT fusion gene is a heterologous reporter construct in which the 5'-flanking sequence and part of the structural region of an oat phytochrome gene are fused to chloramphenicol acetyltransferase (CAT). It is used to measure light-regulated transcriptional activity of the oat phy promoter after transient introduction into plant cells.

SpyTag/SpyCatcher interaction is a protein-domain interaction used as an assistance strategy to improve split fluorescent proteins. In the cited 2019 Communications Biology study, it was demonstrated alongside directed evolution as an approach for improving split sfCherry-based reporters.