Toolkit Items

Focused ultrasound (FUS) is a noninvasive physical delivery and control modality that penetrates deep biological tissues and induces confined mild hyperthermia to activate heat-sensitive genetic modules. In the cited 2023 study, FUS was coupled to heat-sensitive CRISPR, CRISPRa, and CRISPRi systems to enable remote spatiotemporal regulation of genome and epigenome function in live cells and animals.

Single-cell RNA sequencing (scRNA-seq) is a transcriptomic assay method that measures RNA molecules in individual cells by sequencing-based transcript detection. In the cited application, it detected FLiCRE transcripts within the endogenous transcriptome, enabling simultaneous readout of cell type and calcium activation history.

Adeno-associated virus (AAV) is a viral delivery harness used to package and express CRISPR genome-editing components in vivo. In the cited literature, AAV supports single-vector delivery when smaller Cas9 orthologues and their chimeric guide RNAs fit within AAV packaging constraints, enabling robust in vivo genome editing.

Chimeric antigen receptor natural killer (CAR-NK) cells represent a promising "off-the-shelf" alternative to CAR-T cells.

Next-generation CAR designs, such as cytokine-armed CAR-T cells, may enhance T cell infiltration and persistence despite the suppressive TME.

Subsequently, we delve into cutting-edge applications of nanoparticles to enhance immune protection, including mosaic and cocktail nanoparticle vaccines, surface-modified targeting strategies, and the integration of mRNA technology with virus-like particles (VLPs).

This review examines recent advancements in nanoparticle( s) (NPs) delivery systems, with a focus on ... lipid nanoparticles (LNPs)... We discussed various NP platforms and their applications, such as ... dry powder formulations of mRNA-loaded LNPs for pulmonary delivery, and LNP-mediated siRNA delivery for respiratory infections.

Deep brain stimulation (DBS) is an established neuromodulation method used as an add-on treatment for severe Parkinson's disease and other chronic neurological conditions. In the cited review, DBS is presented primarily as the clinical benchmark for comparison with optogenetic neuromodulation.

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.

Here, the latest research progresses in AAV-mediated gene editing and silencing strategies to modify that the genetic ocular diseases are systematically outlined, especially by base editing and prime editing.

CARmacrophages (CAR-M) ... not only phagocytose tumor cells and present antigens but also remodel the immunosuppressive tumor microenvironment

Key innovations in engineered NK cell therapies-including CAR-NK, cytokine armoring (e.g., IL-15), and bispecific/trispecific NK cell engagers (NKCEs)-are critically evaluated.

CRISPR/Cas9 is a bacterial type II genome editing system repurposed as a programmable nuclease for target DNA cleavage and site-specific genome modification. The supplied evidence states that it was engineered for gene editing in mammalian cells by 2013 and is used to interrupt gene expression through cleavage of target DNA.

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.

Three-dimensional (3D) bioprinting is a rapidly evolving technology that uses complementary biomaterials to emulate native extracellular matrices, enabling the generation of finely patterned, multicellular tissue architectures.

Various CRISPR delivery systems, including viral vectors, nanocarriers, and extracellular vesicles, play crucial roles in the effective access of this tool to neural cells.

Non-invasive brain stimulation (NIBS) techniques, such as ... transcranial ultrasound stimulation (TUS), have emerged as promising alternatives.

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.

Translational titration is an application of genetic code expansion in Bacillus subtilis that modulates protein production at the level of translation. In the cited study, it was implemented within a broad and efficient noncanonical amino acid incorporation platform that also supported click-labelling and photo-crosslinking.

functional magnetic resonance imaging (MRI) methods such as diffusion-weighted imaging

The review delves into ongoing efforts in preclinical models, translational advancements, and emerging approaches such as dual-targeting CARs, armored CARs, and alternative co-stimulatory domains.

The review delves into ongoing efforts in preclinical models, translational advancements, and emerging approaches such as dual-targeting CARs, armored CARs, and alternative co-stimulatory domains.

non-viral systems such as lipid nanoparticles and engineered exosomes offer lower toxicity and modularity but face targeting limitations

Exosomes possess antigens and immunostimulatory molecules and can serve as cell-free vaccines to induce antitumor immunity. In addition, given their stability, low immunogenicity, and targeting ability, exosomes represent ideal drug delivery systems in tumor immunotherapy.

Emerging delivery vehicles (AAVs, LNPs, lentivirus, virus-like particles) and their translational implications are discussed.

Lipid-polymer hybrid nanoparticles (LPHNPs) are the next-generation nanocarriers that integrate the mechanical strength and sustained-release capacity of polymeric cores with the biocompatibility and high drug-loading efficiency of lipid shells.

To expand the potential use of contrast-enhanced ultrasound beyond intravascular applications, sub-micron agents, often called nanobubbles or ultra-fine bubbles, have recently emerged as a promising tool.

Light-sheet microscopy, also termed single plane illumination microscopy, is an in vivo fluorescence imaging method tailored to larval research and embryonic imaging. The supplied evidence indicates that it can capture the full course of embryonic development from egg to larva and has been coupled with optogenetic perturbation to study Wnt signaling during embryogenesis.

increasing clinical experience with in-vivo editing - particularly using lipid nanoparticle (LNP) and adeno-associated virus (AAV)-based platforms - that has also revealed important safety considerations

LOVdeg is an engineered protein tag that is appended to a protein of interest to enable blue-light-inducible degradation in Escherichia coli. It provides optically controlled post-translational regulation by coupling light exposure to loss of the tagged protein.

The blue-light-activated DNA template ON switch is a light-responsive DNA construct that enables transcription to be initiated by blue light. In the reported combined system, it was used with a separate light-controlled OFF switch to start transcription with one wavelength and later halt translation of the corresponding mRNA-to-protein output with a different wavelength.

Click-labelling in this context is a Bacillus subtilis genetic code expansion platform that incorporates noncanonical amino acids for click-chemistry-based protein labelling. In the cited 2021 study, it was implemented within broad and efficient stop-codon suppression systems and used alongside photo-crosslinking and translational titration applications.

Photo-crosslinking in this context is an application of genetic code expansion in Bacillus subtilis that enables light-triggered covalent capture of molecular interactions. The reported system was part of a broader noncanonical amino acid incorporation platform used for photo-crosslinking, click-labelling, and translational titration.

Upstream open reading frames (uORFs) are endogenous 5′-leader RNA elements that can take precedence over translation of the main ORF and reduce protein output. In Arabidopsis, blue light can increase use of downstream transcription start sites that bypass uORFs, enabling higher expression of light-responsive genes.

Here, we utilise a combination of lipid-shelled microbubbles (MBs) and ultrasound (US) to physically disperse the biofilm from the growth surface.

Genetic code expansion in Bacillus subtilis is a translation-engineering method for site-specific incorporation of non-standard amino acids into proteins in vivo. The reported system achieved broad and efficient incorporation of 20 distinct non-standard amino acids in B. subtilis using three families of genetic code expansion systems and two codon choices.

Adeno-associated virus (AAV) gene therapy is a promising approach for hemophilia, offering the potential for sustained therapeutic expression of coagulation factors.

Artificial Intelligence (AI)-have emerged as transformative tools to accelerate nanocarrier design and optimise their properties... AI-driven models accelerate the discovery of lipid-based nanoparticle formulations by analysing vast chemical datasets and predicting optimal structures for gene delivery and vaccine development

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.

This review focuses on blood cell membrane-derived nanocarriers as drug delivery and immune-regenerative platforms.

functional magnetic resonance imaging (MRI) methods such as diffusion-weighted imaging, blood oxygen level-dependent MRI

CaRTRIDGE is a mammalian synthetic biology framework that repurposes CRISPR-associated proteins as translational modulators. In this system, Cas proteins repress or activate translation of mRNAs carrying a cognate Cas-binding RNA motif in the 5′ untranslated region, and the platform can be combined with other Cas-based regulatory layers.

Cell-free systems (CFSs) have become powerful tools in synthetic biology, enabling the creation of fast, modular, and customizable biosensors without relying on living cells.

This chapter explores the principles, platforms, and applications of CFS-based HTS... Altogether, CFS-based HTS offers a flexible, rapid, and accessible approach for next-generation biomolecular screening and therapeutic development.

Chimeric antigen receptor natural killer (CAR-NK) cells exert dual cytotoxic effects against tumor cells through CAR-mediated antigen-specific recognition in concert with the nonspecific cytolytic activity mediated by intrinsic NK receptors.

Key innovations in engineered NK cell therapies-including CAR-NK, cytokine armoring (e.g., IL-15), and bispecific/trispecific NK cell engagers (NKCEs)-are critically evaluated.

We concentrate on three principal bioengineered platforms: (3) drug-eluting and biodegradable stents that convert passive luminal scaffolds into active, long-term drug-releasing devices

Engineered bacteriophages are emerging as a promising class of precision antimicrobials... Advances in synthetic biology and nanotechnology have made it possible to redesign phages with enhanced specificity, expanded functionality, and improved stability, positioning them as versatile tools for microbiota-centered therapies.

Engineered virus-like particles (eVLPs) have emerged as a promising class of delivery systems for genome editing agents.

Exo-nanomaterials, hybrids that fuse EV membranes with synthetic cores, aim to unite EV biocompatibility and trafficking with the loading capacity, modularity and stimulus-responsiveness of engineered nanomaterials.