Programmable RNA-guided DNA endonucleases are widespread in eukaryotes and their viruses.

Programmable RNA-guided DNA nucleases perform numerous roles in prokaryotes, but the extent of their spread outside prokaryotes is unclear. Fanzors, the eukaryotic homolog of prokaryotic TnpB proteins, have been detected in genomes of eukaryotes and large viruses, but their activity and functions in eukaryotes remain unknown. Here, we characterize Fanzors as RNA-programmable DNA endonucleases, using biochemical and cellular evidence. We found diverse Fanzors that frequently associate with various eukaryotic transposases. Reconstruction of Fanzors evolution revealed multiple radiations of RuvC-containing TnpB homologs in eukaryotes. Fanzor genes captured introns and proteins acquired nuclear localization signals, indicating extensive, long-term adaptation to functioning in eukaryotic cells. Fanzor nucleases contain a rearranged catalytic site of the RuvC domain, similar to a distinct subset of TnpBs, and lack collateral cleavage activity. We demonstrate that Fanzors can be harnessed for genome editing in human cells, highlighting the potential of these widespread eukaryotic RNA-guided nucleases for biotechnology applications.

Engineering synthetic phosphorylation signaling networks in human cells.

Protein phosphorylation signaling networks play a central role in how cells sense and respond to their environment. Here, we describe the engineering of artificial phosphorylation networks in which "push-pull" motifs-reversible enzymatic phosphorylation cycles consisting of opposing kinase and phosphatase activities-are assembled from modular protein domain parts and then wired together to create synthetic phosphorylation circuits in human cells. We demonstrate that the composability of our design scheme enables model-guided tuning of circuit function and the ability to make diverse network connections; synthetic phosphorylation circuits can be coupled to upstream cell surface receptors to enable fast-timescale sensing of extracellular ligands, while downstream connections can regulate gene expression. We leverage these capabilities to engineer cell-based cytokine controllers that dynamically sense and suppress activated T cells. Our work introduces a generalizable approach for designing and building phosphorylation signaling circuits that enable user-defined sense-and-respond function for diverse biosensing and therapeutic applications.

Programmable RNA-guided endonucleases are widespread in eukaryotes and their viruses.

TnpB proteins are RNA-guided nucleases that are broadly associated with IS200/605 family transposons in prokaryotes. TnpB homologs, named Fanzors, have been detected in genomes of some eukaryotes and large viruses, but their activity and functions in eukaryotes remain unknown. We searched genomes of diverse eukaryotes and their viruses for TnpB homologs and identified numerous putative RNA-guided nucleases that are often associated with various transposases, suggesting they are encoded in mobile genetic elements. Reconstruction of the evolution of these nucleases, which we rename Horizontally-transferred Eukaryotic RNA-guided Mobile Element Systems (HERMES), revealed multiple acquisitions of TnpBs by eukaryotes and subsequent diversification. In their adaptation and spread in eukaryotes, HERMES proteins acquired nuclear localization signals, and genes captured introns, indicating extensive, long term adaptation to functioning in eukaryotic cells. Biochemical and cellular evidence show that HERMES employ non-coding RNAs encoded adjacent to the nuclease for RNA-guided cleavage of double-stranded DNA. HERMES nucleases contain a re-arranged catalytic site of the RuvC domain, similar to a distinct subset of TnpBs, and lack collateral cleavage activity. We demonstrate that HERMES can be harnessed for genome editing in human cells, highlighting the potential of these widespread eukaryotic RNA-guided nucleases for biotechnology applications.

Hierarchical Network-Based Tracklets Data Association for Multiple Extended Target Tracking with Intermittent Measurements.

The key issue of multiple extended target tracking is to differentiate the origins of the measurements. The association of measurements with the possible origins within the target's extent is difficult, especially for occlusions or detection blind zones, which cause intermittent measurements. To solve this problem, a hierarchical network-based tracklet data association algorithm (ET-HT) is proposed. At the low association level, a min-cost network flow model based on the divided measurement sets is built to extract the possible tracklets. At the high association level, these tracklets are further associated with the final trajectories. The association is formulated as an integral programming problem for finding the maximum a posterior probability in the network flow model based on the tracklets. Moreover, the state of the extended target is calculated using the in-coordinate interval Kalman smoother. Simulation and experimental results show the superiority of the proposed ET-HT algorithm over JPDA- and RFS-based methods when measurements are intermittently unavailable.

Force-Mediated Endocytosis of Iron Oxide Nanoparticles for Magnetic Targeting of Stem Cells.

Stem cell therapy represents one of the most promising approaches for tissue repair and regeneration. However, the full potential of stem cell therapy remains to be realized. One major challenge is the insufficient homing and retention of stem cells at the desired sites after in vivo delivery. Here, we provide a proof-of-principle demonstration of magnetic targeting and retention of human muscle-derived stem cells (hMDSCs) in vitro through magnetic force-mediated internalization of magnetic iron oxide nanoparticles (MIONs) and the use of a micropatterned magnet. We found that the magnetic force-mediated cellular uptake of MIONs occurs through an endocytic pathway, and the MIONs were exclusively localized in the lysosomes. The intracellular MIONs had no detrimental effect on the proliferation of hMDSCs or their multilineage differentiation, and no MIONs were translocated to other cells in a coculture system. Using hMDSCs and three other cell types including human umbilical vein endothelial cells (HUVECs), human dermal fibroblasts (HDFs), and HeLa cells, we further discovered that the magnetic force-mediated MION uptake increased with MION size and decreased with cell membrane tension. We found that the cellular uptake rate was initially increased with MION concentration in solution and approached saturation. These findings provide important insight and guidance for magnetic targeting of stem cells in therapeutic applications.

Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases.

Programmable genome integration of large, diverse DNA cargo without DNA repair of exposed DNA double-strand breaks remains an unsolved challenge in genome editing. We present programmable addition via site-specific targeting elements (PASTE), which uses a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of desired payloads. We demonstrate integration of sequences as large as ~36 kilobases at multiple genomic loci across three human cell lines, primary T cells and non-dividing primary human hepatocytes. To augment PASTE, we discovered 25,614 serine integrases and cognate attachment sites from metagenomes and engineered orthologs with higher activity and shorter recognition sequences for efficient programmable integration. PASTE has editing efficiencies similar to or exceeding those of homology-directed repair and non-homologous end joining-based methods, with activity in non-dividing cells and in vivo with fewer detectable off-target events. PASTE expands the capabilities of genome editing by allowing large, multiplexed gene insertion without reliance on DNA repair pathways.

Programmable eukaryotic protein synthesis with RNA sensors by harnessing ADAR.

Programmable approaches to sense and respond to the presence of specific RNAs in biological systems have broad applications in research, diagnostics, and therapeutics. Here we engineer a programmable RNA-sensing technology, reprogrammable ADAR sensors (RADARS), which harnesses RNA editing by adenosine deaminases acting on RNA (ADAR) to gate translation of a cargo protein by the presence of endogenous RNA transcripts. Introduction of a stop codon in a guide upstream of the cargo makes translation contingent on binding of an endogenous transcript to the guide, leading to ADAR editing of the stop codon and allowing translational readthrough. Through systematic sensor engineering, we achieve 277 fold improvement in sensor activation and engineer RADARS with diverse cargo proteins, including luciferases, fluorescent proteins, recombinases, and caspases, enabling detection sensitivity on endogenous transcripts expressed at levels as low as 13 transcripts per million. We show that RADARS are functional as either expressed DNA or synthetic mRNA and with either exogenous or endogenous ADAR. We apply RADARS in multiple contexts, including tracking transcriptional states, RNA-sensing-induced cell death, cell-type identification, and control of synthetic mRNA translation.

RNA-triggered protein cleavage and cell growth arrest by the type III-E CRISPR nuclease-protease.

The type III-E CRISPR-Cas7-11 effector binds a CRISPR RNA (crRNA) and the putative protease Csx29 and catalyzes crRNA-guided RNA cleavage. We report cryo-electron microscopy structures of the Cas7-11-crRNA-Csx29 complex with and without target RNA (tgRNA), and demonstrate that tgRNA binding induces conformational changes in Csx29. Biochemical experiments revealed tgRNA-dependent cleavage of the accessory protein Csx30 by Csx29. Reconstitution of the system in bacteria showed that Csx30 cleavage yields toxic protein fragments that cause growth arrest, which is regulated by Csx31. Csx30 binds Csx31 and the associated sigma factor RpoE (RNA polymerase, extracytoplasmic E), suggesting that Csx30-mediated RpoE inhibition modulates the cellular response to infection. We engineered the Cas7-11-Csx29-Csx30 system for programmable RNA sensing in mammalian cells. Overall, the Cas7-11-Csx29 effector is an RNA-dependent nuclease-protease.

Multifunctional Magnetic Nanoclusters Can Induce Immunogenic Cell Death and Suppress Tumor Recurrence and Metastasis.

Metastasis is the predominant cause of cancer deaths due to solid organ malignancies; however, anticancer drugs are not effective in treating metastatic cancer. Here we report a nanotherapeutic approach that combines magnetic nanocluster-based hyperthermia and free radical generation with an immune checkpoint blockade (ICB) for effective suppression of both primary and secondary tumors. We attached 2,2'-azobis(2-midinopropane) dihydrochloride (AAPH) molecules to magnetic iron oxide nanoclusters (IONCs) to form an IONC-AAPH nanoplatform. The IONC can generate a high level of localized heat under an alternating magnetic field (AMF), which decomposes the AAPH on the cluster surface and produces a large number of carbon-centered free radicals. A combination of localized heating and free radicals can effectively kill tumor cells under both normoxic and hypoxic conditions. The tumor cell death caused by the combination of magnetic heating and free radicals led to the release or exposure of various damage-associated molecule patterns, which promoted the maturation of dendritic cells. Treating the tumor-bearing mice with IONC-AAPH under AMF not only eradicated the tumors but also generated systemic antitumor immune responses. The combination of IONC-AAPH under AMF with anti-PD-1 ICB dramatically suppressed the growth of untreated distant tumors and induced long-term immune memory. This IONC-AAPH based magneto-immunotherapy has the potential to effectively combat metastasis and control cancer recurrence.

Subsecond multichannel magnetic control of select neural circuits in freely moving flies.

Precisely timed activation of genetically targeted cells is a powerful tool for the study of neural circuits and control of cell-based therapies. Magnetic control of cell activity, or 'magnetogenetics', using magnetic nanoparticle heating of temperature-sensitive ion channels enables remote, non-invasive activation of neurons for deep-tissue applications and freely behaving animal studies. However, the in vivo response time of thermal magnetogenetics is currently tens of seconds, which prevents precise temporal modulation of neural activity. Moreover, magnetogenetics has yet to achieve in vivo multiplexed stimulation of different groups of neurons. Here we produce subsecond behavioural responses in Drosophila melanogaster by combining magnetic nanoparticles with a rate-sensitive thermoreceptor (TRPA1-A). Furthermore, by tuning magnetic nanoparticles to respond to different magnetic field strengths and frequencies, we achieve subsecond, multichannel stimulation. These results bring magnetogenetics closer to the temporal resolution and multiplexed stimulation possible with optogenetics while maintaining the minimal invasiveness and deep-tissue stimulation possible only by magnetic control.

Magnetic Iron Oxide Nanoparticles for Biomedical Applications.

Owing to the quantum confinement at the nanoscale, magnetic iron oxide nanoparticles (MIONs) consisting of magnetite and maghemite nanocrystals have unique physical properties, enabling a wide range of biomedical applications by utilizing mechanical, magnetic, chemical, and thermal effects of MIONs respectively. For example, MIONs can serve as a contrast agent for magnetic resonance imaging (MRI), convert electromagnetic energy into thermal energy for hyperthermia therapy, and carry drug/gene for targeted in vivo delivery. In this review, we discuss the recent development of MION based engineering approaches and their biomedical applications, including sensitive protein quantification, magnetic nanoparticle heating, in vivo molecular imaging, and drug delivery. The opportunities and challenges in further exploring the biomedical applications of MIONs are also briefly discussed.

Preparation and laser sintering of a thermoplastic polyurethane carbon nanotube composite-based pressure sensor.

Selective laser sintering (SLS) is a desirable method for fabricating human motion detecting sensors as it can produce a complex shape with different materials that are machinable to specific applications. The bottleneck in the SLS processing of sensors is the preparation of a material that is both flexible and conductive. In this study, carbon nanotubes (CNTs) were selected as a conductive nanofiller and dispersed into a flexible thermoplastic polyurethane (TPU) polymer matrix to prepare TPU/CNT composites for SLS processing pressure sensors. CNTs were first oxidized to prevent them from aggregating in the TPU matrix. TPU/CNT composites were prepared via solution blending and ball milling methods, and the dispersion of the CNTs in the composites was observed by scanning electron microscopy. The thermal properties of TPU/CNT composites with different CNT content were measured, and processing parameters used in the SLS were determined based on differential scanning calorimetry measurements. SLS-processed TPU/CNT composites were prepared with different conductivity and piezoresistive properties. Percolation theory and piezoresistive performance results proved that a 0.25 wt% CNT-containing TPU/CNT composite showed the best pressure sensing ability, and it was successfully used as a sensor to detect plantar pressure distribution in a human foot.

Study on the Characteristics of Walnut Shell/Co-PES/Co-PA Powder Produced by Selective Laser Sintering.

Agricultural and forestry wastes are used as materials for selective laser sintering (SLS) to alleviate resource shortage, reduce the pollution of the environment, lower the cost of materials, and improve the accuracy of parts produced by SLS. However, the mechanical properties of wood⁻plastic parts are poor, and thus they cannot be applied widely. In order to improve the mechanical properties of wood⁻plastic parts, a new type of walnut shell polymer composite (WSPC) was prepared by a polymer mixing method and was used to produce parts via SLS. Additionally, the dimensional accuracy, morphologies, density, and mechanical properties of the WSPC parts were studied. The results showed that the addition of a small amount of copolyamide (Co-PA) powder could effectively improve the mechanical properties and decrease the density of the WSPC parts. By increasing the amount of Co-PA powder and decreasing that of copolyester (Co-PES) powder, the mechanical properties first increased, then decreased, and finally increased again; in addition, the density first decreased then increased. By increasing the preheating temperature, the mechanical properties and density of the WSPC parts were enhanced.

Study on the Ingredient Proportions and After-Treatment of Laser Sintering Walnut Shell Composites.

To alleviate resource shortage, reduce the cost of materials consumption and the pollution of agricultural and forestry waste, walnut shell composites (WSPC) consisting of walnut shell as additive and copolyester hot melt adhesive (Co-PES) as binder was developed as the feedstock of selective laser sintering (SLS). WSPC parts with different ingredient proportions were fabricated by SLS and processed through after-treatment technology. The density, mechanical properties and surface quality of WSPC parts before and after post processing were analyzed via formula method, mechanical test and scanning electron microscopy (SEM), respectively. Results show that, when the volume fraction of the walnut shell powder in the WSPC reaches the maximum (40%), sintered WSPC parts have the smallest warping deformation and the highest dimension precision, although the surface quality, density, and mechanical properties are low. However, performing permeating resin as the after-treatment technology could considerably increase the tensile, bending and impact strength by 496%, 464%, and 516%, respectively.