Directed Evolution

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.

Time-resolved vibrational spectroscopy coupled with isotope labeling is an assay method used to resolve light-triggered structural dynamics in the Avena sativa LOV2 (AsLOV2) photosensory domain. In the cited study, it mapped structural evolution from 100 fs to 1 ms after optical excitation and supported a sequential allosteric model linking the flavin pocket to Jα-helix unfolding.

TMT-opsins are vertebrate nonvisual photopigments identified in brain interneurons and motorneurons, including cells co-expressing VAL-opsins. Reported expression patterns and isolated adult tectal slice physiology associate TMT-opsin-expressing neuronal populations with intrinsic light responsiveness in the fish brain.

VAL-opsins are vertebrate nonvisual photopigments identified in brain tissue and studied in the context of photosensory interneurons and motorneurons. The cited evidence links VAL-opsin-positive neuronal populations to light-responsive tectal interneurons and to co-expression with TMT-opsins in distinct interneurons and motorneurons.

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.

Fluorescent protein-based reporters and biosensors are presented as optical tools for observing signaling states relevant to neuromodulatory GPCR pathways.

The high dose/refuge approach is an insect resistance management strategy used with Bt crops to combat the evolution of insect resistance. In the supplied evidence, it is identified as one of the two main IRM strategies used in the U.S.

Light-induction hardware-software platforms are optogenetic delivery systems that provide controlled illumination using formats ranging from simple illumination set-ups to microscopy, microtiter plate, and bioreactor designs. They are used to stimulate biological systems with light, and automated implementations can support computer-controlled experiments with in silico feedback control.

Microscopy designs for optogenetic stimulation are light-delivery platform configurations that range from simple illumination set-ups to sophisticated microscopy, microtiter plate, and bioreactor designs. These platforms support optogenetic stimulation across experimental scales from single-cell stimulation to whole-culture illumination, and some incorporate automated measurement and stimulation for computer-controlled operation.

Microtiter plate designs for optogenetic stimulation are light-delivery platform formats used to apply regulated optical inputs in biological experiments. The cited review places these designs within a broader range of optogenetic hardware spanning simple illumination setups to microtiter plate and bioreactor implementations, with use cases from single-cell stimulation to whole-culture illumination.

Photopharmacology is an engineering method that uses chemically engineered small-molecule photosensitive effectors to impose light control over biological targets. In the cited literature, it is identified as one of the major strategies for targeted photocontrol in proteins and as an optical approach for modulating adrenergic receptor signaling with temporal and spatial precision.

Prime editing is a versatile "search-and-replace" genome-editing technology that enables precise and flexible genome correction of genetic sequences by reverse-transcribing an RNA template encoded at the 3' end of a prime editing guide RNA (pegRNA).

AlphaFold3 is a computational structure-prediction method used in the cited study to model the MagMboI–DNA complex. In that work, it was applied to infer interactions with the 5'-GATC-3' recognition sequence and to guide optimization of the photoactivatable endonuclease variant MagMboI-plus for top-down genome engineering.

macrophages ... establishing CAR-engineered macrophages (CAR-M) as a highly promising next-generation therapeutic platform

PAR3/INSC/LGN is an evolutionarily conserved multi-protein complex that forms a polarity-associated machinery required for asymmetric cell division in the developing brain. Human genetic evidence further links this machinery to function in the adult peripheral nervous system, where its dysfunction is associated with tubulin aggregation and necrotic neurodegeneration.

Split APEX peroxidase (sAPEX) is an engineered two-fragment version of APEX generated by directed evolution. It consists of a 200-amino acid N-terminal fragment (AP) and a 50-amino acid C-terminal fragment (EX) that can reconstitute peroxidase activity in mammalian cells.

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.

Countermeasures now integrate synergistic Cry/Vip pyramiding

Optogenetics has revolutionized the field of neuroscience by enabling precise control of neural activity through light-sensitive proteins known as opsins.

Light inducible dimer pairs are multicomponent optogenetic switches derived from cyanobacteriochrome photoswitchable proteins. They enable light-dependent heterodimerization and were developed to support orthogonal control of biological processes with red, green, and blue light.

LOV-Turbo is a light-regulated proximity-labeling system generated by installing a light-sensitive LOV domain into the biotin ligase TurboID. It provides rapid and reversible control of biotin-labeling activity with low-power blue light and can also be activated by luciferase-derived BRET for interaction-dependent proximity labeling in living cells.

LOV-based optogenetic tools are multi-component light-responsive systems built from Light-Oxygen-Voltage sensing domains. These domains function as versatile photoreceptors involved in cellular signaling and environmental adaptation across kingdoms of life, and their continued development is intended to expand optical control of biological systems.

Here, we introduce AdaptUC, a computational framework that demonstrates how the fraction of biomass precursors synthesized from unadapted carbon sources governs both the evolutionary driving force and the minimal substrate requirement.

Recent breakthroughs in cryo-electron microscopy (cryo-EM) and artificial intelligence (AI)-based structure prediction have revolutionized protein modeling by enabling highly accurate computational predictions from amino acid sequences.

The resulting degron system, named AID 2.1, maintains effective target protein depletion with minimal basal degradation and faster recovery after ligand washout, enabling characterization and rescue experiments for essential genes.

To address these limitations, we employ base-editing-mediated mutagenesis followed by several rounds of functional selection and screening. This directed protein evolution generates several gain-of-function OsTIR1 variants, including S210A, that significantly enhance the overall degron efficiency.

Computational tools such as Cello have further revolutionized TFB design, enabling in silico optimization and construction of complex genetic circuits for integrating multiple signals and achieving precise gene regulation.

The title and supplied source summary explicitly indicate coverage of adoptive cellular therapy and CAR-T / chimeric antigen receptor approaches.

This review comprehensively examines the evolution and current landscape of RNA therapeutics, encompassing major classes such as mRNA vaccines, small interfering RNAs (siRNAs), antisense oligonucleotides (ASOs), and emerging RNA editing technologies like CRISPR-Cas13.

Recent advances in synthetic biology, particularly the development of CRISPR-Cas genome editing technology, offer a revolutionary approach to designing Lactobacillus strains with customized traits... we reviewed the research progress of CRISPR-Cas9 gene editing in Lactobacillus... CRISPR-Cas9 toolkits are expected to achieve directed evolution of strain performance.

Countermeasures now integrate ... CRISPR/Cas9-validated receptor knockouts revealing functional redundancy

Recent breakthroughs in cryo-electron microscopy (cryo-EM) and artificial intelligence (AI)-based structure prediction have revolutionized protein modeling by enabling near-atomic resolution visualization.

Engineered chimeric receptors are described as an opsin-related optical tool class for manipulating neuromodulatory GPCR signaling. They imply receptor constructs that couple light input to GPCR pathway control.

The supplied web research summary identifies EvoMining as an evolutionary genome-mining approach that complements mainstream biosynthetic gene cluster search tools and supports discovery of hidden biosynthetic chemistry.

Opsin-free optogenetics has emerged as a powerful tool in cancer immunotherapy because its versatility and photoactivation kinetics align with the timescale of immune cell signaling, and it has given rise to the subfield of optogenetic immunoengineering.

we developed SyMetrics, a framework that integrates predictors of splicing, RNA stability, evolutionary conservation, codon usage, synonymous variation effects, sequence properties, and allele frequency

This review explores the revolutionary integration of synNotch systems with CAR T-cell therapy... This study highlights the potential of synNotch-based CAR-T cells to transform the field of targeted cancer therapy by resolving present challenges.