On-Site Formation of Functional Dopaminergic Presynaptic Terminals on Neuroligin-2-Modified Gold-Coated Microspheres.

Advancements in neural interface technologies have enabled the direct connection of neurons and electronics, facilitating chemical communication between neural systems and external devices. One promising approach is a synaptogenesis-involving method, which offers an opportunity for synaptic signaling between these systems. Janus synapses, one type of synaptic interface utilizing synaptic cell adhesion molecules for interface construction, possess unique features that enable the determination of location, direction of signal flow, and types of neurotransmitters involved, promoting directional and multifaceted communication. This study presents the first successful establishment of a Janus synapse between dopaminergic (DA) neurons and abiotic substrates by using a neuroligin-2 (NLG2)-mediated synapse-inducing method. NLG2 immobilized on gold-coated microspheres can induce synaptogenesis upon contact with spatially isolated DA axons. The induced DA Janus synapses exhibit stable synaptic activities comparable to that of native synapses over time, suggesting their suitability for application in neural interfaces. By calling for DA presynaptic organizations, the NLG2-immobilized abiotic substrate is a promising tool for the on-site detection of synaptic dopamine release.

Streamlining the interface between electronics and neural systems for bidirectional electrochemical communication.

Seamless neural interfaces conjoining neurons and electrochemical devices hold great potential for highly efficient signal transmission across neural systems and the external world. Signal transmission through chemical sensing and stimulation via electrochemistry is remarkable because communication occurs through the same chemical language of neurons. Emerging strategies based on synaptic interfaces, iontronics-based neuromodulation, and improvements in selective neurosensing techniques have been explored to achieve seamless integration and efficient neuro-electronics communication. Synaptic interfaces can directly exchange signals to and from neurons, in a similar manner to that of chemical synapses. Hydrogel-based iontronic chemical delivery devices are operationally compatible with neural systems for improved neuromodulation. In this perspective, we explore developments to improve the interface between neurons and electrodes by targeting neurons or sub-neuronal regions including synapses. Furthermore, recent progress in electrochemical neurosensing and iontronics-based chemical delivery is examined.

Neuroligin-1-Modified Electrodes for Specific Coupling with a Presynaptic Neuronal Membrane.

Coordination of synapses onto electrodes with high specificity and maintaining a stable and long-lasting interface have importance in the field of neural interfaces. One potential approach is to present ligands on the surface of electrodes that would be bound through a protein-protein interaction to specific areas of neuronal cells. Here, we functionalize electrode surfaces with genetically engineered neuroligin-1 protein and demonstrate the formation of a nascent presynaptic bouton upon binding to neurexin-1 ß on the presynaptic membrane of neurons. The resulting synaptically connected electrode shows an assembly of presynaptic proteins and comparable exocytosis kinetics to that of native synapses. Importantly, a neuroligin-1-induced synapse-electrode interface exhibits type specificity and structural robustness. We envision that the use of synaptic adhesion proteins in modified neural electrodes may lead to new approaches in the interfacing of neural circuity and electronics.

Robust Induced Presynapse on Artificial Substrates as a Neural Interfacing Method.

Over the recent years, the development of neural interface systems has stuck to using electrical cues to stimulate neurons and read out neural signals, although neurons relay signals via chemical release and recognition at synapses. In addition, conventional neural interfaces are vulnerable to cell migration and glial encapsulation due to the absence of connection anchoring the neuron into the device unlike synapses, which are firmly sustained by protein bonding. To close this discrepancy, we conducted an intensive investigation into the induced synapse interface by employing engineered synaptic proteins from a neural interface perspective. The strong potential of induced synaptic differentiation as an emerging neural interfacing technique is demonstrated by exploring its structural features, chemical release kinetics, robustness, and scalability to the brain tissue. We show that the exocytosis kinetics of induced synapses is similar to that of endogenous synapses. Moreover, induced synapses show remarkable stability, despite cell migration and growth. The synapse-inducing technique has broad applications to cultured hippocampal and cortex tissues and suggests a promising method to integrate neural circuits with digital elements.

A miniaturized solid salt reverse electrodialysis battery: a durable and fully ionic power source.

A novel pump-free miniaturized reverse electrodialysis (RED) system was designed to provide lasting power transduced from salinity gradients, named solid salt RED (ssRED), and this quasi-battery uses a solid salt instead of electrolyte solution for streamlined usage. It is portable, flexible, comparable in size to a universal serial bus flash drive, and easily activated with a small amount of water. It maintains a constant ionic concentration gradient through precipitation reactions between a pair of different salts. This precipitation-assisted solid salt RED (PssRED) is an unprecedented ionic power source as it can generate steady electricity in the absence of a driving pump. The PssRED was successfully coupled with bipolar electrode (BPE) microchip sensors which require stable ionic electricity and a polyelectrolyte ionic diode to realize a fully ionic circuit. It is envisioned that the range of application could be expanded to supply electromotive force to various devices through an ionic charge flow.

Miniaturized Reverse Electrodialysis-Powered Biosensor Using Electrochemiluminescence on Bipolar Electrode.

We suggest an electrochemiluminescence (ECL)-sensing platform driven by ecofriendly, disposable, and miniaturized reverse electrodialysis (RED) patches as an electric power source. The flexible RED patches composed of ion-exchange membranes (IEMs) can produce voltage required for ECL sensing by simply choosing the appropriate number of IEMs and the ratio of salt concentrations. We integrate the RED patch with a bipolar electrode on the microfluidic chip to demonstrate the proof-of-concept, i.e., glucose detection in the range of 0.5-10 mM by observing ECL emissions with naked eyes. The miniaturized RED-powered biosensing system is widely applicable for electrochemical-sensing platforms. This is expected to be a solution for practical availability of battery-free electrochemical sensors for disease diagnosis in developing countries.

Genome-wide target specificities of CRISPR RNA-guided programmable deaminases.

Cas9-linked deaminases, also called base editors, enable targeted mutation of single nucleotides in eukaryotic genomes. However, their off-target activity is largely unknown. Here we modify digested-genome sequencing (Digenome-seq) to assess the specificity of a programmable deaminase composed of a Cas9 nickase (nCas9) and the deaminase APOBEC1 in the human genome. Genomic DNA is treated with the base editor and a mixture of DNA-modifying enzymes in vitro to produce DNA double-strand breaks (DSBs) at uracil-containing sites. Off-target sites are then computationally identified from whole genome sequencing data. Testing seven different single guide RNAs (sgRNAs), we find that the rAPOBEC1-nCas9 base editor is highly specific, inducing cytosine-to-uracil conversions at only 18 ± 9 sites in the human genome for each sgRNA. Digenome-seq is sensitive enough to capture off-target sites with a substitution frequency of 0.1%. Notably, off-target sites of the base editors are often different from those of Cas9 alone, calling for independent assessment of their genome-wide specificities.

Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells.

Programmable clustered regularly interspaced short palindromic repeats (CRISPR) Cpf1 endonucleases are single-RNA-guided (crRNA) enzymes that recognize thymidine-rich protospacer-adjacent motif (PAM) sequences and produce cohesive double-stranded breaks (DSBs). Genome editing with CRISPR-Cpf1 endonucleases could provide an alternative to CRISPR-Cas9 endonucleases, but the determinants of targeting specificity are not well understood. Using mismatched crRNAs we found that Cpf1 could tolerate single or double mismatches in the 3' PAM-distal region, but not in the 5' PAM-proximal region. Genome-wide analysis of cleavage sites in vitro for eight Cpf1 nucleases using Digenome-seq revealed that there were 6 (LbCpf1) and 12 (AsCpf1) cleavage sites per crRNA in the human genome, fewer than are present for Cas9 nucleases (>90). Most Cpf1 off-target cleavage sites did not produce mutations in cells. We found mismatches in either the 3' PAM-distal region or in the PAM sequence of 12 off-target sites that were validated in vivo. Off-target effects were completely abrogated by using preassembled, recombinant Cpf1 ribonucleoproteins.