Clinical significance of the genetically variable landscape of the gut microbiome in patients with gestational diabetes mellitus patients.

Background: The composition of the gut microbiome has been recorted to be strongly associated with gestational diabetes mellitus (GDM), but mutational characterization of the microbiome in patients with GDM has been overlooked. Here, we revealed the genetic variation landscape of the gut microbiome and assessed its clinical significance in a cohort of patients with GDM.

Methods: We employed a macrogenomic dataset made up of a discovery cohort of 54 cases and a validation cohort of 220 cases to screen for high-abundance microbial flora and identified single nucleotide variants (SNVs) and insertions/deletions (indels).

Trends in developing one-pot CRISPR diagnostics strategies.

The integration of nucleic acid amplification (NAA) with the CRISPR detection system has led to significant advancements and opportunities for development in molecular diagnostics. Nevertheless, the incompatibility between CRISPR cleavage and NAA has significantly impeded the commercialization of this technology. Currently, several one-pot detection strategies based on CRISPR systems have been devised to address concerns regarding aerosol contamination risk and operational complexity associated with step-by-step detection as well as the sensitivity limitation of conventional one-pot methods.

Light-Start CRISPR-Cas12a Reaction with Caged crRNA Enables Rapid and Sensitive Nucleic Acid Detection.

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a promising platform for nucleic acid detection. Regulating the CRISPR reaction would be extremely useful to improve the detection efficiency and speed of CRISPR diagnostic applications. Here, we have developed a light-start CRISPR-Cas12a reaction by employing caged CRISPR RNA (crRNA).

Development of a Handheld Nano-centrifugal Device for Visual Virus Detection.

Unlabelled: Gold nanoparticles (AuNPs) colorimetric assays based on distance-dependent optical characteristics have been widely employed for bioanalysis. However, this assay is not effective for visually detecting low-concentration targets due to the faint color change. Here, we developed a handheld nano-centrifugal device which could separate the crosslinked and non-crosslinked AuNPs.

Photocontrolled crRNA activation enables robust CRISPR-Cas12a diagnostics.

CRISPR diagnostics based on nucleic acid amplification faces barriers to its commercial use, such as contamination risks and insufficient sensitivity. Here, we propose a robust solution involving optochemical control of CRISPR RNA (crRNA) activation in CRISPR detection. Based on this strategy, recombinase polymerase amplification (RPA) and CRISPR-Cas12a detection systems can be integrated into a completely closed test tube.

Fast microwave heating-based one-step synthesis of DNA and RNA modified gold nanoparticles.

DNA/RNA-gold nanoparticle (DNA/RNA-AuNP) nanoprobes have been widely employed for nanobiotechnology applications. Here, we discover that both thiolated and non-thiolated DNA/RNA can be efficiently attached to AuNPs to achieve high-stable spherical nucleic acid (SNA) within minutes under a domestic microwave (MW)-assisted heating-dry circumstance. Further studies show that for non-thiolated DNA/RNA the conjugation is poly (T/U) tag dependent.

M-CDC: Magnetic pull-down-assisted colorimetric method based on the CRISPR/Cas12a system.

The construction of a rapid, simple, and specific nucleic acid detection platform is of great significance to the control of the large-scale spread of infectious diseases. We have recently established a magnetic pull-down-assisted colorimetric method based on the CRISPR/Cas12a system (termed M-CDC), which effectively integrates the advantages of CRISPR/Cas12a, magnetic beads-based separation, and AuNP bioprobe to provide a simple and specific biosensing platform for nucleic acid assay. The M-CDC method is compatible with point-of-care testing and enables the detection of nucleic acid samples in less than an hour without relying on expensive and complex instruments.

Detection of SARS-CoV-2 by CRISPR/Cas12a-Enhanced Colorimetry.

The outbreak of COVID-19 caused a worldwide public health crisis. Large-scale population screening is an effective means to control the spread of COVID-19. Reverse transcription-polymerase chain reaction (RT-qPCR) and serology assays are the most available techniques for SARS-CoV-2 detection; however, they suffer from either less sensitivity and accuracy or low instrument accessibility for screening.

Simultaneous Dual-Gene Diagnosis of SARS-CoV-2 Based on CRISPR/Cas9-Mediated Lateral Flow Assay.

Few methods for the detection of SARS-CoV-2 currently have the capability to simultaneously detect two genes in a single test, which is a key measure to improve detection accuracy, as adopted by the gold standard RT-qPCR method. Developed here is a CRISPR/Cas9-mediated triple-line lateral flow assay (TL-LFA) combined with multiplex reverse transcription-recombinase polymerase amplification (RT-RPA) for rapid and simultaneous dual-gene detection of SARS-CoV-2 in a single strip test. This assay is characterized by the detection of envelope (E) and open reading frame 1ab (Orf1ab) genes from cell-cultured SARS-CoV-2 and SARS-CoV-2 viral RNA standards, showing a sensitivity of 100 RNA copies per reaction (25 μL).

Single-Step, Salt-Aging-Free, and Thiol-Free Freezing Construction of AuNP-Based Bioprobes for Advancing CRISPR-Based Diagnostics.

The recently reported freezing-based labeling method for constructing DNA-AuNP probes is rapid but still requires thiol modification. Here, we evaluated a poly(A)-tagged DNA sequence using the freezing-based labeling method, and the results demonstrated that approximately 10 A bases at the sequence ends are essential. More detailed observations revealed that some DNA sequences tend to form secondary structures and thus shield exposed A bases, resulting in inefficient or failed labeling.

Universal and Naked-Eye Gene Detection Platform Based on the Clustered Regularly Interspaced Short Palindromic Repeats/Cas12a/13a System.

Gold-nanoparticles-based colorimetric assay is an attractive detection format, but is limited by the tedious and ineffective posthybridization manipulations for genomic analysis. Here, we present a new design for a colorimetric gene-sensing platform based on the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system. In this strategy, programmable recognition of DNA by Cas12a/crRNA and RNA by Cas13a/crRNA with a complementary target activates the -ssDNA or -ssRNA cleavage.

The crystal structure of KSHV ORF57 reveals dimeric active sites important for protein stability and function.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a γ-herpesvirus closely associated with Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman disease. Open reading frame 57 (ORF57), a viral early protein of KSHV promotes splicing, stability and translation of viral mRNA and is essential for viral lytic replication. Previous studies demonstrated that dimerization of ORF57 stabilizes the protein, which is critical for its function.