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Cell-free biology is the activation of biological processes without the use of intact living cells. It has been used for more than 50 years across the life sciences as a foundational research tool, but a recent technical renaissance has facilitated high-yielding (grams of protein per litre), cell-free gene expression systems from model bacteria, the development of cell-free platforms from non-model organisms and multiplexed strategies for rapidly assessing biological design. These advances provide exciting opportunities to profoundly transform synthetic biology by enabling new approaches to the model-driven design of synthetic gene networks, the fast and portable sensing of compounds, on-demand biomanufacturing, building cells from the bottom up, and next-generation educational kits.Cell-free gene expression systems have long been used to address fundamental research questions. Now, owing to technological advances, these systems are finding wider applications in the field of synthetic biology, including in biosensing, biomanufacturing, education and the design of gene networks.

Integrating synthetic biology into wearables could expand opportunities for noninvasive monitoring of physiological status, disease states and exposure to pathogens or toxins. However, the operation of synthetic circuits generally requires the presence of living, engineered bacteria, which has limited their application in wearables. Here we report lightweight, flexible substrates and textiles functionalized with freeze-dried, cell-free synthetic circuits, including CRISPR-based tools, that detect metabolites, chemicals and pathogen nucleic acid signatures. The wearable devices are activated upon rehydration from aqueous exposure events and report the presence of specific molecular targets by colorimetric changes or via an optical fiber network that detects fluorescent and luminescent outputs. The detection limits for nucleic acids rival current laboratory methods such as quantitative PCR. We demonstrate the development of a face mask with a lyophilized CRISPR sensor for wearable, noninvasive detection of SARS-CoV-2 at room temperature within 90 min, requiring no user intervention other than the press of a button. Wearable materials are endowed with synthetic biology circuits to detect biomolecules, including SARS-CoV-2 RNA.

The recent outbreak of novel coronavirus (SARS-CoV-2) causing COVID-19 disease spreads rapidly in the world. Rapid and early detection of SARS-CoV-2 facilitates early intervention and prevents the disease spread. Here, we present an All-In-One Dual CRISPR-Cas12a (AIOD-CRISPR) assay for one-pot, ultrasensitive, and visual SARS-CoV-2 detection. By targeting SARS-CoV-2’s nucleoprotein gene, two CRISPR RNAs without protospacer adjacent motif (PAM) site limitation are introduced to develop the AIOD-CRISPR assay and detect the nucleic acids with a sensitivity of few copies. We validate the assay by using COVID-19 clinical swab samples and obtain consistent results with RT-PCR assay. Furthermore, a low-cost hand warmer (~$0.3) is used as an incubator of the AIOD-CRISPR assay to detect clinical samples within 20 min, enabling an instrument-free, visual SARS-CoV-2 detection at the point of care. Thus, our method has the significant potential to provide a rapid, sensitive, one-pot point-of-care assay for SARS-CoV-2. Rapid and early detection of SARS-CoV-2 will aid intervention to stop disease spread. Here the authors present a one-pot CRISPR-based rapid detection system with visual readout.

The blood proteome holds great promise for precision medicine but poses substantial challenges due to the low abundance of most plasma proteins and the vast dynamic range of the plasma proteome. Here we address these challenges with NUcleic acid Linked Immuno-Sandwich Assay (NULISA™), which improves the sensitivity of traditional proximity ligation assays by ~10,000-fold to attomolar level, by suppressing assay background via a dual capture and release mechanism built into oligonucleotide-conjugated antibodies. Highly multiplexed quantification of both low- and high-abundance proteins spanning a wide dynamic range is achieved by attenuating signals from abundant targets with unconjugated antibodies and next-generation sequencing of barcoded reporter DNA. A 200-plex NULISA containing 124 cytokines and chemokines and other proteins demonstrates superior sensitivity to a proximity extension assay in detecting biologically important low-abundance biomarkers in patients with autoimmune diseases and COVID-19. Fully automated NULISA makes broad and in-depth proteomic analysis easily accessible for research and diagnostic applications. Unlocking the blood proteome requires exquisite sensitivity and multiplexing to detect low and high abundance proteins simultaneously. Here the authors describe a 200-plex immunoassay with attomolar sensitivity to detect important low abundance proteins in inflammatory diseases and COVID-19.

Although polymerase chain reaction (PCR) is the most widely used method for DNA amplification, the requirement of thermocycling limits its non-laboratory applications. Isothermal DNA amplification techniques are hence valuable for on-site diagnostic applications in place of traditional PCR. Here we describe a true isothermal approach for amplifying and detecting double-stranded DNA based on a CRISPR–Cas9-triggered nicking endonuclease-mediated Strand Displacement Amplification method (namely CRISDA). CRISDA takes advantage of the high sensitivity/specificity and unique conformational rearrangements of CRISPR effectors in recognizing the target DNA. In combination with a peptide nucleic acid (PNA) invasion-mediated endpoint measurement, the method exhibits attomolar sensitivity and single-nucleotide specificity in detection of various DNA targets under a complex sample background. Additionally, by integrating the technique with a Cas9-mediated target enrichment approach, CRISDA exhibits sub-attomolar sensitivity. In summary, CRISDA is a powerful isothermal tool for ultrasensitive and specific detection of nucleic acids in point-of-care diagnostics and field analyses. Isothermal DNA amplification techniques are useful for diagnostic applications in place of traditional PCR. Here the authors describe CRISDA, which combines CRISPR–Cas9 with strand displacement amplification and exhibits attomolar sensitivity and single-nucleotide specificity in DNA detection.

Exosomes and extracellular vesicles (EV) are increasingly being explored as circulating biomarkers, but their heterogenous composition will likely mandate the development of multiplexed EV technologies. Iteratively multiplexed analyses of near single EVs have been challenging to implement beyond a few colors during spectral sensing. Here we developed a multiplexed analysis of EV technique (MASEV) to interrogate thousands of individual EVs during 5 cycles of multi-channel fluorescence staining for 15 EV biomarkers. Contrary to the common belief, we show that: several markers proposed to be ubiquitous are less prevalent than believed; multiple biomarkers concur in single vesicles but only in small fractions; affinity purification can lead to loss of rare EV subtypes; and deep profiling allows detailed analysis of EV, potentially improving the diagnostic content. These findings establish the potential of MASEV for uncovering fundamental EV biology and heterogeneity and increasing diagnostic specificity. Multiplexed analyses of near single EVs is currently challenging. Here the authors report the method MASEV, multiplexed analysis of EVs, to interrogate thousands of individual EVs during 5 cycles of multi-channel fluorescence staining for 15 EV biomarkers.

Nucleic acid detection powered by CRISPR technology provides a rapid, sensitive, and deployable approach to molecular diagnostics. While exciting, there remain challenges limiting its practical applications, such as the need for pre-amplification and the lack of quantitative ability. Here, we develop an asymmetric CRISPR assay for cascade signal amplification detection of nucleic acids by leveraging the asymmetric trans -cleavage behavior of competitive crRNA. We discover that the competitive reaction between a full-sized crRNA and split crRNA for CRISPR-Cas12a can induce cascade signal amplification, significantly improving the target detection signal. In addition, we find that CRISPR-Cas12a can recognize fragmented RNA/DNA targets, enabling direct RNA detection by Cas12a. Based on these findings, we apply our asymmetric CRISPR assay to quantitatively detect microRNA without the need for pre-amplification, achieving a detection sensitivity of 856 aM. Moreover, using this method, we analyze and quantify miR-19a biomarker in plasma samples from bladder cancer patients. This asymmetric CRISPR assay has the potential to be widely applied for simple and sensitive nucleic acid detection in various diagnostic settings. New strategies are being developed to simplify CRISPR-based nucleic acid detection. By investigating the competitive reaction between a full-sized crRNA and split crRNA for CRISPR-Cas12a, the authors develop an asymmetric CRISPR assay for amplification-free, cascade signal amplification detection of nucleic acids.

The CRISPR-Cas12a RNA-guided complexes have tremendous potential for nucleic acid detection but are limited to the picomolar detection limit without an amplification step. Here, we develop a platform with engineered crRNAs and optimized conditions that enabled us to detect various clinically relevant nucleic acid targets with higher sensitivity, achieving a limit of detection in the femtomolar range without any target pre-amplification step. By extending the 3′- or 5′-ends of the crRNA with different lengths of ssDNA, ssRNA, and phosphorothioate ssDNA, we discover a self-catalytic behavior and an augmented rate of LbCas12a-mediated collateral cleavage activity as high as 3.5-fold compared to the wild-type crRNA and with significant improvement in specificity for target recognition. Particularly, the 7-mer DNA extension to crRNA is determined to be universal and spacer-independent for enhancing the sensitivity and specificity of LbCas12a-mediated nucleic acid detection. We perform a detailed characterization of our engineered ENHANCE system with various crRNA modifications, target types, reporters, and divalent cations. With isothermal amplification of SARS-CoV-2 RNA using RT-LAMP, the modified crRNAs are incorporated in a paper-based lateral flow assay that can detect the target with up to 23-fold higher sensitivity within 40–60 min. CRISPR-Cas12a based detection systems can be sensitive down to the picomolar range. Here the authors modify the 3′- and 5′-ends of the crRNA and show this enhances trans-cleavage for improved sensitivity.

Bioprinting promises enormous control over the spatial deposition of cells in three dimensions 1 – 7 , but current approaches have had limited success at reproducing the intricate micro-architecture, cell-type diversity and function of native tissues formed through cellular self-organization. We introduce a three-dimensional bioprinting concept that uses organoid-forming stem cells as building blocks that can be deposited directly into extracellular matrices conducive to spontaneous self-organization. By controlling the geometry and cellular density, we generated centimetre-scale tissues that comprise self-organized features such as lumens, branched vasculature and tubular intestinal epithelia with in vivo-like crypts and villus domains. Supporting cells were deposited to modulate morphogenesis in space and time, and different epithelial cells were printed sequentially to mimic the organ boundaries present in the gastrointestinal tract. We thus show how biofabrication and organoid technology can be merged to control tissue self-organization from millimetre to centimetre scales, opening new avenues for drug discovery, diagnostics and regenerative medicine. A 3D bioprinting approach has been developed to facilitate tissue morphogenesis by directly depositing organoid-forming stem cells in an extracellular matrix, with the ability to generate intestinal epithelia and branched vascular tissue constructs.

The COVID-19 pandemic has highlighted that new diagnostic technologies are essential for controlling disease transmission. Here, we develop SHINE (Streamlined Highlighting of Infections to Navigate Epidemics), a sensitive and specific diagnostic tool that can detect SARS-CoV-2 RNA from unextracted samples. We identify the optimal conditions to allow RPA-based amplification and Cas13-based detection to occur in a single step, simplifying assay preparation and reducing run-time. We improve HUDSON to rapidly inactivate viruses in nasopharyngeal swabs and saliva in 10 min. SHINE’s results can be visualized with an in-tube fluorescent readout — reducing contamination risk as amplification reaction tubes remain sealed — and interpreted by a companion smartphone application. We validate SHINE on 50 nasopharyngeal patient samples, demonstrating 90% sensitivity and 100% specificity compared to RT-qPCR with a sample-to-answer time of 50 min. SHINE has the potential to be used outside of hospitals and clinical laboratories, greatly enhancing diagnostic capabilities. The COVID-19 pandemic has highlighted the need for user-friendly diagnostic techniques. Here, the authors present SHINE, a streamlined and optimised Cas13-based method with accompanying smartphone app for visual diagnosis.