Engineered CRISPRa System for Precise and Multiplex Gene Regulation Using a Hybrid dCas12a Variant and Hairpin-Spacer crRNAs.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a nucleases have emerged as a promising alternative to CRISPR-Cas9 in gene editing and expression regulation. However, the adoption of Cas12a has been hindered due to general off-target activities and limited efficiency. Here, we utilized a hybrid engineered Cas12a variant and hairpin-spacer crRNAs (h-CAP) to enhance the specificity and efficiency of the CRISPR-Cas12a system. Leveraging the h-CAP strategy, we demonstrate both single-base-specific and multiplex gene expression regulation in human cells.

Profiling extracellular vesicle surface proteins with 10 µL peripheral plasma within 4 h.

Extracellular vesicle (EV) surface proteins, expressed by primary tumours, are important biomarkers for early cancer diagnosis. However, the detection of these EV proteins is complicated by their low abundance and interference from non-EV components in clinical samples. Herein, we present a MEmbrane-Specific Separation and two-step Cascade AmpLificatioN (MESS2CAN) strategy for direct detection of EV surface proteins within 4 h. MESS2CAN utilises novel lipid probes (long chains linked by PEG2K with biotin at one end, and DSPE at the other end) and streptavidin-coated magnetic beads, permitting a 49.6% EV recovery rate within 1 h. A dual amplification strategy with a primer exchange reaction (PER) cascaded by the Cas12a system then allows sensitive detection of the target protein at 10 EV particles per microliter. Using 4 cell lines and 90 clinical test samples, we demonstrate MESS2CAN for analysing HER2, EpCAM and EGFR expression on EVs derived from cells and patient plasma. MESS2CAN reports the desired specificity and sensitivity of EGFR (AUC = 0.98) and of HER2 (AUC = 1) for discriminating between HER2-positive breast cancer, triple-negative breast cancer and healthy donors. MESS2CAN is a pioneering method for highly sensitive in vitro EV diagnostics, applicable to clinical samples with trace amounts of EVs.

One-Pot Visual Detection of African Swine Fever Virus Using CRISPR-Cas12a.

African swine fever virus (ASFV) is a leading cause of worldwide agricultural loss. ASFV is a highly contagious and lethal disease for both domestic and wild pigs, which has brought enormous economic losses to a number of countries. Conventional methods, such as general polymerase chain reaction and isothermal amplification, are time-consuming, instrument-dependent, and unsatisfactorily accurate. Therefore, rapid, sensitive, and field-deployable detection of ASFV is important for disease surveillance and control. Herein, we created a one-pot visual detection system for ASFV with CRISPR/Cas12a technology combined with LAMP or RPA. A mineral oil sealing strategy was adopted to mitigate sample cross-contamination between parallel vials during high-throughput testing. Furthermore, the blue fluorescence signal produced by ssDNA reporter could be observed by the naked eye without any dedicated instrument. For CRISPR-RPA system, detection could be completed within 40 min with advantageous sensitivity. While CRISPR-LAMP system could complete it within 60 min with a high sensitivity of 5.8 × 102 copies/µl. Furthermore, we verified such detection platforms display no cross-reactivity with other porcine DNA or RNA viruses. Both CRISPR-RPA and CRISPR-LAMP systems permit highly rapid, sensitive, specific, and low-cost Cas12a-mediated visual diagnostic of ASFV for point-of-care testing (POCT) applications.

Harnessing Multiplex crRNA in the CRISPR/Cas12a System Enables an Amplification-Free DNA Diagnostic Platform for ASFV Detection.

CRISPR-associated (Cas) protein systems have been increasingly incorporated in nucleic-acid diagnosis. CRISPR/Cas12a can cleave single-stranded DNA (ssDNA) after being guided to the target double-stranded DNA (dsDNA) with crRNA, making it a specific tool for dsDNA detection. Assisted by nucleic acid preamplification, CRISPR/Cas12a enables dsDNA detection at the attomolar level. However, such mandatory preamplification in CRISPR/Cas12a also accompanies the extra step of transferring preamplification products into the CRISPR/Cas12a system, which is not only cumbersome and time-consuming but also induces the risk of cross-contamination. Herein, we demonstrate a multiplex-crRNA strategy to enhance the sensitivity of the CRISPR/Cas12a system without any preamplification. This multiplex-crRNA strategy harnesses multiple sequences of crRNA which target different regions of the same dsDNA substrate in the same CRISPR/Cas12a system. Therefore, detection signals are accumulated without amplification, which augments the conventional detection limit. For application demonstration, the B646L gene from the African swine fever virus (ASFV), which is a dsDNA virus, is exemplified. The detection limit of the multiplex-crRNA system can be improved to ∼1 picomolar (pM) without amplification, which is ∼64 times stronger than the conventional single-crRNA system. The multiplex-crRNA system presented in this study, with slight modifications, can be generalized to other biosensing settings where preamplification is not readily available.

2'-O-Methyl modified guide RNA promotes the single nucleotide polymorphism (SNP) discrimination ability of CRISPR-Cas12a systems.

The CRISPR-Cas12a system has been widely applied to genome editing and molecular diagnostics. However, off-target cleavages and false-positive results remain as major concerns in Cas12a practical applications. Herein, we propose a strategy by utilizing the 2'-O-methyl (2'-OMe) modified guide RNA (gRNA) to promote the Cas12a's specificity. Gibbs free energy analysis demonstrates that the 2'-OMe modifications at the 3'-end of gRNA effectively suppress the Cas12a's overall non-specific affinity while maintaining high on-target affinity. For general application illustrations, HBV genotyping and SARS-CoV-2 D614G mutant biosensing platforms are developed to validate the enhanced Cas12a's specificity. Our results indicate that the 2'-OMe modified gRNAs could discriminate single-base mutations with at least two-fold enhanced specificity compared to unmodified gRNAs. Furthermore, we investigate the enhancing mechanisms of the 2'-OMe modified Cas12a systems by molecular docking simulations and the results suggest that the 2'-OMe modifications at the 3'-end of gRNA reduce the Cas12a's binding activity to off-target DNA. This work offers a versatile and universal gRNA design strategy for highly specific Cas12a system development.

Encountering and Wrestling: Neutrophils Recognize and Defensively Degrade Graphene Oxide.

The boosting exploitation of graphene oxide (GO) increases exposure risk to human beings. However, as primary defender in the first immune line, neutrophils' mechanism of defensive behavior toward GO remains unclear. Herein, we discovered that neutrophils recognize and defensively degrade GO in a lateral dimension dependent manner. The micrometer-sized GO (mGO) induces NETosis by releasing neutrophil extracellular traps (NETs), while nanometer-sized GO (nGO) elicits neutrophil degranulation. The two neutrophils' defensive behaviors are accompanied with generation of reactive oxygen species and activation of p-ERK and p-Akt kinases. However, mGO-induced NETosis is NADPH oxidase (NOX)-independent while nGO-triggered degranulation is NOX-dependent. Furthermore, myeloperoxidase (MPO) is determinant mediator despite distinct neutrophil phenotypes. Neutrophils release NETs comprising of MPO upon activated with mGO, while MPO is secreted via nGO-induced degranulation. Moreover, the binding energy between MPO and GO is calculated to be 69.8728 kJ mol-1 , indicating that electrostatic interactions mainly cause the spontaneous binding process. Meanwhile, the central enzymatic biodegradation occurs at oxygenic active sites and defects on GO. Mass spectrometry analysis deciphers the degradation products are biocompatible molecules like flavonoids and polyphenols. This study provides fundamental evidence and practical guidance for nanotechnology based on GO, including vaccine adjuvant, implantable devices, and energy storage.

CRISPR/Cas12a Powered DNA Framework-Supported Electrochemical Biosensing Platform for Ultrasensitive Nucleic Acid Analysis.

Nucleic acid analysis using ultrasensitive and simple methods is critically important for the early-stage diagnosis and treatment of diseases. The CRISPR/Cas proteins, guided by a single-stranded RNA have shown incredible capability for sequence-specific targeting and detection. Herein, in order to improve and expand the application of CRISPR/Cas technology to the electrochemical interface-based nucleic acids analysis, the authors develop a CRISPR/Cas12a powered DNA framework-supported electrochemical biosensing platform via the cis and trans cleavage of Cas12a on the heterogeneous carbon interface (the existing publications which commonly adopted trans-cleavage). Their solid-liquid interface is first immobilized by 3D tetrahedral framework nucleic acids (FNAs) with specific DNA recognition probe. Based on the recognition of the complementary target through protospacer adjacent motif (PAM) confirmation and CRISPR-derived RNA (crRNA) matching, the easily formed Cas12a/crRNA duplex can get access to the interface, and the cis and trans cleavage of Cas12a can be easily activated. In combination with the enzyme catalyzed reaction, they achieved an ultralow limit of detection (LOD) of 100 fm in HPV-16 detection without pre-amplification. Furthermore, the platform is compatible with a spike-in human serum sample and has superior stability. Thus, their reported platform offers a practical, versatile, and amplification-free toolbox for ultrasensitive nucleic acid analysis.

One-pot pre-coated interface proximity extension assay for ultrasensitive co-detection of anti-SARS-CoV-2 antibodies and viral RNA.

In the field of in vitro diagnostics, detection of nucleic acids and proteins from biological samples is typically performed with independent platforms; however, co-detection remains a major technical challenge. Specifically, during the coronavirus disease 2019 (COVID-19) pandemic, the ability to simultaneously detect viral RNA and human antibodies would prove highly useful for efficient diagnosis and disease course management. Herein, we present a multiplex one-pot pre-coated interface proximity extension (OPIPE) assay that facilitates the simultaneous recognition of antibodies using a pre-coated antigen interface and a pair of anti-antibodies labeled with oligonucleotides. Following anti-antibody-bound nucleic acid chain extension to form templates in proximity, antibody signals can be amplified, together with that of targeted RNA, via a reverse transcription-polymerase chain reaction. Using four-color fluorescent TaqMan probes, we demonstrate the co-detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibodies and viral nucleic acids in a single bio-complex sample, including nucleocapsid protein-specific IgG and IgM, and the RNA fragments of RdRp and E genes. The serum detection limit for this platform is 100 fg/mL (0.67 fM) for the anti-SARS-CoV-2 antibody and 10 copies/µL for viral RNA. The OPIPE assay offers a practical and affordable solution for ultrasensitive co-detection of nucleic acids and antibodies from the same trace biological sample without the additional requirement of complicated equipment.

A HiPAD Integrated with rGO/MWCNTs Nano-Circuit Heater for Visual Point-of-Care Testing of SARS-CoV-2.

Nowadays, the main obstacle for further miniaturization and integration of nucleic acids point-of-care testing devices is the lack of low-cost and high-performance heating materials for supporting reliable nucleic acids amplification. Herein, reduced graphene oxide hybridized multi-walled carbon nanotubes nano-circuit integrated into an ingenious paper-based heater is developed, which is integrated into a paper-based analytical device (named HiPAD). The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still raging across the world. As a proof of concept, the HiPAD is utilized to visually detect the SARS-CoV-2 N gene using colored loop-mediated isothermal amplification reaction. This HiPAD costing a few dollars has comparable detection performance to traditional nucleic acids amplifier costing thousands of dollars. The detection range is from 25 to 2.5 × 1010 copies mL-1 in 45 min. The detection limit of 25 copies mL-1 is 40 times more sensitive than 1000 copies mL-1 in conventional real-time PCR instruments. The disposable paper-based chip could also avoid potential secondary transmission of COVID-19 by convenient incineration to guarantee biosafety. The HiPAD or easily expanded M-HiPAD (for multiplex detection) has great potential for pathogen diagnostics in resource-limited settings.

Single-Cell Microwell Platform Reveals Circulating Neural Cells as a Clinical Indicator for Patients with Blood-Brain Barrier Breakdown.

Central nervous system diseases commonly occur with the destruction of the blood-brain barrier. As a primary cause of morbidity and mortality, stroke remains unpredictable and lacks cellular biomarkers that accurately quantify its occurrence and development. Here, we identify NeuN+/CD45-/DAPI+ phenotype nonblood cells in the peripheral blood of mice subjected to middle cerebral artery occlusion (MCAO) and stroke patients. Since NeuN is a specific marker of neural cells, we term these newly identified cells as circulating neural cells (CNCs). We find that the enumeration of CNCs in the blood is significantly associated with the severity of brain damage in MCAO mice (p < 0.05). Meanwhile, the number of CNCs is significantly higher in stroke patients than in negative subjects (p < 0.0001). These findings suggest that the amount of CNCs in circulation may serve as a clinical indicator for the real-time prognosis and progression monitor of the occurrence and development of ischemic stroke and other nervous system disease.

New Structure Mass Tag based on Zr-NMOF for Multiparameter and Sensitive Single-Cell Interrogating in Mass Cytometry.

Mass cytometry, also called cytometry by time-of-flight (CyTOF), is an emerging powerful proteomic analysis technique that utilizes metal chelated polymer (MCP) as mass tags for interrogating high-dimensional biomarkers simultaneously on millions of individual cells. However, under the typical polymer-based mass tag system, the sensitivity and multiplexing detection ability has been highly restricted. Herein, a new structure mass tag based on a nanometal organic framework (NMOF) is reported for multiparameter and sensitive single-cell biomarker interrogating in CyTOF. A uniform-sized Zr-NMOF (33 nm) carrying 105 metal ions is synthesized under modulator/reaction time coregulation, which is monodispersed and colloidally stable in water for over one-year storage. On functionalization with an antibody, the Zr mass tag exhibits specific molecular recognition properties and minimal cross-reaction toward nontargeted cells. In addition, the Zr-mass tag is compatible with MCP mass tags in a multiparameter assay for mouse spleen cells staining, which exploits four additional channels, m/z = 90, 91, 92, 94, for single-cell immunoassays in CyTOF. Compared to the MCP mass tag, the Zr-mass tag provides an additional fivefold signal amplification. This work provides the fundamental technical capability for exploiting NMOF-based mass tags for CyTOF application, which opens up possibility of high-dimensional single-cell immune profiling, low abundant antigen detection, and development of new barcoding systems.

Hairpin-Spacer crRNA-Enhanced CRISPR/Cas13a System Promotes the Specificity of Single Nucleotide Polymorphism (SNP) Identification.

The Cas13a system has great potential in RNA interference and molecular diagnostic fields. However, lacking guidelines for crRNA design hinders practical applications of the Cas13a system in RNA editing and single nucleotide polymorphism identification. This study posits that crRNAs with hairpin spacers improve the specificity of CRISPR/Cas13a system (termed hs-CRISPR). Gibbs free energy analysis suggests that the hairpin-spacer crRNAs (hs-crRNAs) suppress Cas13a's affinity to off-target RNA. A hepatitis B virus DNA genotyping platform is established to further validate the high-specificity of hs-CRISPR/Cas13a system. Compared to ordinary crRNA, hs-crRNAs increase the specificity by threefold without sacrificing the sensitivity of the CRISPR/Cas13a system. Furthermore, the mechanism of the Cas13a/hs-crRNA/target RNA composition is elucidated with theoretical simulations. This work builds on the fundamental understanding of Cas13a activation and offers significant improvements for the rational design of crRNA for the CRISPR/Cas13a system.

A One-Pot Toolbox Based on Cas12a/crRNA Enables Rapid Foodborne Pathogen Detection at Attomolar Level.

Bacterial contamination accounts for more than half of food poisoning cases. Conventional methods such as colony-counting and general polymerase chain reaction are time-consuming, instrument-dependent, and sometimes not accurate. Herein, we developed a novel one-pot toolbox with precision and ultra sensitivity (OCTOPUS) platform for foodborne pathogen detection based on the mechanism in which Cas12a nontarget binding unleashes its collateral DNase activity. We demonstrated its application on two widespread foodborne bacteria, namely, E. coli O157:H7 and Streptococcus aureus, using specific crRNA targeting rfbE and nuc gene, respectively. For better sensitivity, recombinase polymerase amplification (RPA) was integrated without product purification. This one-pot detection, that is, RPA reagent, crRNA, and ssDNA-FQ reporter are all in one tube with the subsequent addition of Cas12a enzyme, was able to detect genomic DNA at the attomolar level. It omits an extra cap-opening process to avoid practical inconvenience and possible cross-sample contamination. Moreover, we demonstrated this platform for a real food matrix. A simple water boiling method for genome extraction together with one-pot assay achieved a limit of detection value of 1 CFU/mL in less than 50 min. This OCTOPUS technique integrates bacterial genome extraction, preamplification based on RPA, and Cas12a/crRNA cleavage assay.