CRISPR/Cas13a-Responsive and RNA-Bridged DNA Hydrogel Capillary Sensor for Point-of-Care Detection of RNA.

Disease diagnostics and surveillance increasingly highlight the importance of portable, cost-effective, and sensitive point-of-care (POC) detection of nucleic acids. Here, we report a CRISPR/Cas13a-responsive and RNA-bridged DNA hydrogel capillary sensor for the direct and visual detection of specific RNA with high sensitivity. The capillary sensor was simply prepared by loading RNA-cross-linking DNA hydrogel film (∼0.2 mm ± 0.02 mm) at the end of a capillary. When CRISPR/Cas13a specifically recognizes the target RNA, the RNA bridge in the hydrogel film is cleaved by the trans-cleavage activity of CRISPR/Cas13a, increasing the permeability of the hydrogel film. Different concentrations of target RNA activate different amounts of Cas13a, cleaving different amounts of the RNA bridge in the hydrogel and causing corresponding changes in the permeability of the hydrogel. Therefore, samples containing different amounts of the target RNA travel to different distances in the capillary. Visual reading of the distance provides quantitative detection of the RNA target without the need for any nucleic acid amplification or auxiliary equipment. The technique was successfully used for the determination of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in clinical nasopharyngeal (NP) swab and saliva samples. Easily quantifiable distance using a ruler eliminates the need for any optical or electrochemical detection equipment, making this assay potentially useful for POC and on-site applications.

CRISPR-Cas-Driven Single Micromotor (Cas-DSM) Enables Direct Detection of Nucleic Acid Biomarkers at the Single-Molecule Level.

The target-dependent endonuclease activity (also known as the trans-cleavage activity) of CRISPR-Cas systems has stimulated great interest in the development of nascent sensing strategies for nucleic acid diagnostics. Despite many attempts, the majority of the sensitive CRISPR-Cas diagnostics strategies mainly rely on nucleic acid preamplification, which generally needs complex probes/primers designs, multiple experimental steps, and a longer testing time, as well as introducing the risk of false-positive results. In this work, we propose the CRISPR-Cas-Driven Single Micromotor (Cas-DSM), which can directly detect the nucleic acid targets at a single-molecule level with high specificity. We have demonstrated that the Cas-DSM is a reliable and practical method for the quantitative detection of DNA/RNA in various complex clinical samples as well as in individual cells without any preamplification processes. Due to the excellent features of the CRISPR/Cas system, including constant temperature, simple design, high specificity, and flexible programmability, the Cas-DSM could serve as a simple and universal platform for nucleic acid detection. More importantly, this work will provide a breakthrough for the development of next-generation amplification-free CRISPR/Cas sensing toolboxes.

Maternal plasma diacylglycerols and triacylglycerols in the prediction of gestational diabetes mellitus.

OBJECTIVE: To define the lipidomic profile in plasma across pregnancy, and identify lipid biomarkers for gestational diabetes mellitus (GDM) prediction in early pregnancy. DESIGN: Case-control study. SETTING: Tertiary referral maternity unit. POPULATION OR SAMPLE: Plasma samples from 100 GDM and 100 normal glucose tolerance (NGT) women, divided into a training set (GDM first trimester = 50, GDM second trimester = 40, NGT first trimester = 50, NGT second trimester = 50) and a validation set (GDM first trimester = 45, GDM second trimester = 34, NGT first trimester = 44, NGT second trimester = 40). METHODS: Plasma samples were collected in the first (11+0 to 13+6 weeks), second (19+0 to 24+6 weeks), and third trimesters (30+0 to 34+6 weeks), and tested by ultra-high-performance liquid chromatography coupled with electrospray ionisation-quadrupole-time of flight-mass spectrometry; The GDM prediction model was established by the machine-learning method of random forest. MAIN OUTCOME MEASURES: Gestational diabetes mellitus. RESULTS: In both the GDM and NGT group, lyso-glycerophospholipids were down-regulated, whereas ceramides, sphingomyelins, cholesteryl ester, diacylglycerols (DGs) and triacylglycerols (TGs) and glucosylceramide were up-regulated across the three trimesters of pregnancy. In the training dataset, seven TGs and five DGs demonstrated good performance in the prediction of GDM in the first and second trimesters (area under the curve [AUC] = 0.96 with 95% confidence interval [CI] of 0.93-1 and AUC = 0.97 with 95% CI of 0.95-1, respectively), independent of maternal body mass index (BMI) and ethnicity. In the validation dataset, the predictive model achieved an AUC of 0.88 and 0.94 at the first and second trimesters, respectively. CONCLUSIONS: Our results have proposed new lipid biomarkers for the first trimester prediction of GDM, independent of ethnicity and BMI.

Specific detection of fusion transcripts based on a duplex-specific nuclease and isothermal exponential amplification reaction.

Fusion genes are mostly found in tumor tissues, but are in low expression levels in healthy tissues, making them good candidate biomarkers for tumor diagnosis and therapy. Here, we propose a duplex-specific nuclease-isothermal exponential amplification reaction (DSN-IEXPAR) method for the detection of fusion transcripts. A DNA probe is specifically designed for fusion transcript recognition and hybridization, and DSN cleavages the DNA probe in the DNA/RNA duplex. Through controlling the recognition and cleavage temperature, DSN can only cut the DNA probe fully matched with the target fusion transcript rather than other transcripts containing partial the same sequence, endowing the proposed method with high specificity to the fusion transcript in the presence of homologous sequences. The truncated DNA probe after cutting can subsequently trigger IEXPAR as a probe, so as low as 100 fM fusion transcript can be detected with the proposed DSN-IEXPAR. The evaluation of the analytical performance of DSN-IEXPAR demonstrates that it can provide an effective platform for fusion transcript detection in the ordinary laboratory and clinical diagnosis.

Sensitive detection of fusion transcripts with padlock probe-based continuous cascade amplification (P-CCA).

Gene fusion, resulting from chromosomal rearrangements, is the juxtaposition of two or more original genes into the same set to form a functional gene. The significant specificity of fusion genes for tumor cells makes them promising candidates for diagnostic biomarkers and therapeutic targets. The sensitive detection of fusion transcripts is of great significance in biological research and disease diagnosis. Here, we propose a method for the sensitive detection of PML-RARα gene fusion transcripts by the direct ligation of the padlock probe at the junction site, and the cyclized DNA then triggers a continuous cascade amplification of two subsequent amplification reactions: rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP). Due to the ability of the ligation reaction to differentiate mismatched sequences and the high amplification efficiency of continuous cascade amplification reactions, the proposed method can detect as low as 1 fM targets with high specificity, and has been successfully applied to real samples. Through a facile design of the triggering sequence in padlock probes, the cascade RCA and LAMP can be integrated into one-tube isothermal reactions with a simple one-step operation. Therefore, this work provides a convenient padlock probe-based continuous cascade amplification (P-CCA) method for the detection of fusion transcripts, and offers a fast and reliable platform for the early clinical diagnosis of gene fusion-related cancers.

Temporal persistence of residual fetal cell-free DNA from a deceased cotwin after selective fetal reduction in dichorionic diamniotic twin pregnancies.

OBJECTIVES: To determine the temporal persistence of the residual cell-free DNA (cfDNA) of the deceased cotwin in maternal circulation after selective fetal reduction and evaluate its long persistence in noninvasive prenatal testing (NIPT). METHODS: Dichorionic diamniotic twins (N = 5) undergoing selective fetal reduction because of a trisomy were recruited. After informed consent, maternal blood was collected immediately before reduction and periodically after reduction until birth. The plasma cfDNA of each sample was sequenced and analyzed for fetal aneuploidy and fetal fractions. RESULTS: In all pregnancies, the fetal fraction of the cfDNA of the deceased fetus increased to peak at 7-9 weeks after fetal reduction, and subsequently decreased gradually to almost undetectable during the late third trimester. The NIPT T-scores persistently reflected the detection of fetal trisomy up to 16 (median 9.5) weeks after fetal reduction. CONCLUSIONS: Residual cfDNA from the deceased cotwin after selective reduction at 14-17 gestational weeks led to the persistent generation of false-positive NIPT results for up to 16 weeks postdemise. Thus, providing NIPT for pregnancies with a cotwin demise in early second trimester is prone to misleading results and not recommended.

The Setup and Application of Reference Material in Sequencing-Based Noninvasive Prenatal Testing.

INTRODUCTION: The sequencing-based noninvasive prenatal testing (NIPT) has been successfully integrated into clinical practice and facilitated the early detection of fetal chromosomal anomalies. However, a comprehensive reference material to evaluate and quality control NIPT services from different NIPT providers remains unavailable. METHODS: In this study, we established a set of NIPT reference material consisting of 192 simulated samples. Most of the potential factors influencing the accuracy of NIPT, such as fetal fraction, mosaicism, and interfering substances, were included in the reference material. We compared the performance of chromosomal abnormalities detection on 3 widely used sequencers (NextSeq 500, BGISEQ-500, and Ion Proton) based on the reference material. RESULTS: All 3 sequencers provided highly accurate and reliable results to samples with ≥3.5% fetal fractions and high percentage of mosaicism. CONCLUSIONS: The established reference material can serve as a universal standard quality control for the current and new-coming NIPT providers based on various sequencers.

Lipidomic Biomarkers of Extracellular Vesicles for the Prediction of Preterm Birth in the Early Second Trimester.

Preterm birth is the leading cause of infant death worldwide and results in a high societal economic burden associated with newborn care. Recent studies have shown that extracellular vesicles (EVs) play an important role in fetal development during pregnancy. Lipids in EVs related to preterm birth remain undefined. Here, we fully investigated differences in lipids in plasma, microvesicles (MVs), and exosomes (Exos) between 27 preterm and 66 full-term pregnant women in the early second trimester (12-24 weeks) using an untargeted lipidomics approach. Independent of other characteristics of samples, we detected 97, 58, and 10 differential features (retention time (RT) and m/z) with identification in plasma, MVs, and Exos, respectively. A panel of five lipids from MVs has an area under the receiver operating characteristic curve (AUC) of 0.87 for the prediction of preterm birth. One lipid of the panel (PS (34:0)) was validated in an additional 83 plasma samples (41 preterm and 42 full-term deliveries) by the pseudotargeted lipidomics method (AUC = 0.71). Our results provide useful information about the early prediction of preterm birth, as well as a better understanding of the underlying mechanisms and intervention of preterm birth. The MS data have been deposited in the CNSA (https://db.cngb.org/cnsa/) of CNGBdb with accession code CNP0001076.

Real-time quantification of fusion transcripts with ligase chain reaction by direct ligation of adjacent DNA probes at fusion junction.

Gene fusions, produced by aberrant juxtapositions of two or more genes even in different chromosomes, play important roles in the primary oncogenic mechanism and have been demonstrated to be typically associated with many cancers. So the fused genes or the transcripts can be specific predictive biomarkers for cancer diagnosis and therapy. Herein, we develop a direct ligation- and ligase chain reaction (LCR)-based method for a fusion transcript assay. In virtue of the high selectivity of ligase and the exponential amplification capacity of LCR, the proposed method can detect as low as 1 fM fusion transcripts with high specificity and has been successfully applied to real samples. With the real-time fluorescence measurements, the fusion transcripts can be assayed in a simple way. Therefore, the proposed method can provide a simple and cost-effective platform for fusion transcript detection in routine laboratories and clinical diagnosis.

Direct recognition and sensitive detection of circular RNA with ligation-based PCR.

Circular RNAs (circRNAs), which function as a kind of newly discovered biomarker, have been shown to play an important role in gene expression, signaling pathway and disease diagnosis. Accurate recognition and sensitive detection of sequence-specific circRNA of interest can advance our understanding of its biological functions and the diagnosis of human diseases. Herein, we have newly developed a highly sensitive and specific method to quantitatively detect circRNA through accurate ligation of two ingeniously designed DNA probes using splintR ligase at the junction site of circRNA, which can directly discriminate circRNA from the corresponding linear RNA. With PCR amplification of the ligated DNA probes, circRNA as low as 1 fM can be detected with the dynamic range spanning over five orders of magnitude. This method has been successfully applied to the detection of circRNA in total RNA samples extracted from cell lines.

Intracellular Messenger RNA Triggered Catalytic Hairpin Assembly for Fluorescence Imaging Guided Photothermal Therapy.

We show a theranostic nanoplatform for messenger RNA (mRNA) triggered enhanced fluorescence imaging guided therapy. Catalytic hairpin assembly (CHA) and gold nanorods (AuNRs) are employed to fabricate the theranostic nanoplatform. Two hairpin DNAs and Cy5 labeled duplex DNA are integrated into the CHA for mRNA triggered fluorescence signal amplification via hybridization and displacement with mRNA. The AuNRs act both as the fluorescence quencher and the photothermal therapy (PTT) agent. The nanoplatform not only enables sensitive and specific imaging of target mRNA in living cells and good differentiating of the survivin mRNA expression levels in different cell lines but also offers excellent photothermal conversion efficiency for PTT. The developed nanoplatform has great potential for sensitive and specific intracellular mRNA imaging guided PTT.

Noninvasive prenatal testing of trisomies 21 and 18 by massively parallel sequencing of maternal plasma DNA in twin pregnancies.

OBJECTIVE: The objective of this study is to assess the performance of noninvasive prenatal testing for trisomies 21 and 18 on the basis of massively parallel sequencing of cell-free DNA from maternal plasma in twin pregnancies. METHOD: A double-blind study was performed over 12 months. A total of 189 pregnant women carrying twins were recruited from seven hospitals. Maternal plasma DNA sequencing was performed to detect trisomies 21 and 18. The fetal karyotype was used as gold standard to estimate the sensitivity and specificity of sequencing-based noninvasive prenatal test. RESULTS: There were nine cases of trisomy 21 and two cases of trisomy 18 confirmed by karyotyping. Plasma DNA sequencing correctly identified nine cases of trisomy 21 and one case of trisomy 18. The discordant case of trisomy 18 was an unusual case of monozygotic twin with discordant fetal karyotype (one normal and the other trisomy 18). The sensitivity and specificity of maternal plasma DNA sequencing for fetal trisomy 21 were both 100% and for fetal trisomy 18 were 50% and 100%, respectively. CONCLUSION: Our study further supported that sequencing-based noninvasive prenatal testing of trisomy 21 in twin pregnancies could be achieved with a high accuracy, which could effectively avoid almost 95% of invasive prenatal diagnosis procedures.

Specific quantitative detection of N6-methyladenosine at single-base resolution by extension-based isothermal exponential amplification reaction (E-IEXPAR).

BACKGROUND: N6-methyladenosine (m6A) is a common modification in RNA, crucial for various cellular functions and associated with human diseases. Quantification of m6A at single-base resolution is of great significance for exploring its biological roles and related disease research. However, existing analysis techniques, such as polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP), face challenges like the requirement for thermal cycling or intricate primer design. Therefore, it is urgent to establish a simple, non-thermal cycling and highly sensitive assay for m6A. RESULTS: Leveraging the inhibitory effect of m6A on primer elongation and uncomplicated feature of the isothermal exponential amplification reaction (IEXPAR), we have developed an extension-based IEXPAR (E-IEXPAR). This approach requires just a single extension primer and one template, simplifying the design process in comparison to the more complex primer requirements of the LAMP methods. The reactions are conducted at constant temperatures, therby elimiating the use of thermal cycling that needed in PCR methods. By combining IEXPAR with an extension reaction, E-IEXPAR can identify m6A in RNA concentrations as low as 4 fM. We have also introduced a new analytical model to process E-IEXPAR results, which can aid to minimize the impact of unmodified adenine (A) on m6A measurements, enabling accurate m6A quantification in small mixed samples and cellular RNA specimens. SIGNIFICANCE AND NOVELTY: E-IEXPAR streamlines m6A detection by eliminating the need for intricate primer design and thermal cycling, which are common in current analytical methods. Its utilization of an extension reaction for the initial identification of m6A, coupled with a novel calculation model tailored to E-IEXPAR outcomes, ensures accurate m6A selectivity in mixed samples. As a result, E-IEXPAR offers a reliable, straightforward, and potentially economical approach for specifically assaying m6A in both biological function studies and clinical research.

Longitudinal integrative cell-free DNA analysis in gestational diabetes mellitus.

Gestational diabetes mellitus (GDM) presents varied manifestations throughout pregnancy and poses a complex clinical challenge. High-depth cell-free DNA (cfDNA) sequencing analysis holds promise in advancing our understanding of GDM pathogenesis and prediction. In 299 women with GDM and 299 matched healthy pregnant women, distinct cfDNA fragment characteristics associated with GDM are identified throughout pregnancy. Integrating cfDNA profiles with lipidomic and single-cell transcriptomic data elucidates functional changes linked to altered lipid metabolism processes in GDM. Transcription start site (TSS) scores in 50 feature genes are used as the cfDNA signature to distinguish GDM cases from controls effectively. Notably, differential coverage of the islet acinar marker gene PRSS1 emerges as a valuable biomarker for GDM. A specialized neural network model is developed, predicting GDM occurrence and validated across two independent cohorts. This research underscores the high-depth cfDNA early prediction and characterization of GDM, offering insights into its molecular underpinnings and potential clinical applications.

Whole-genome DNA methylation in skin lesions from patients with psoriasis vulgaris.

Psoriasis, a chronic inflammatory skin disorder, is characterized by aberrant keratinocyte proliferation and differentiation in the epidermis. Although the pathogenesis of psoriasis is still incompletely understood, both genetic susceptibilities and environmental triggers are known to act as key players in its development. Several studies have suggested that DNA methylation is involved in the pathogenesis of psoriasis. However, the precise mechanisms underlying the regulation and maintenance of the methylome as well as their relationship with this disease remain poorly characterized. Herein, we used methylated DNA immunoprecipitation sequencing (MeDIP-Seq) to characterize whole-genome DNA methylation patterns in involved and uninvolved skin lesions from patients with psoriasis. The results of our MeDIP-Seq analyses identified differentially methylated regions (DMRs) covering almost the entire genome with sufficient depth and high resolution, showing that the number of hypermethylated DMRs was considerably higher than that of hypomethylated DMRs in involved psoriatic skin samples. Moreover, gene ontology analysis of MeDIP-Seq data showed that the aberrantly methylated genes belonged to several different ontological domains, such as the immune system, cell cycle and apoptosis. The results of the bisulfite-sequencing experiments for the genes PDCD5 and TIMP2 confirmed the methylation status identified by MeDIP-Seq, and the mRNA expression levels of these two genes were consistent with their DNA methylation profiles. To our knowledge, the present study constitutes the first report on MeDIP-Seq in psoriasis. The identification of whole-genome DNA methylation patterns associated with psoriasis provides new insight into the pathogenesis of this complex disease and represents a promising avenue through which to investigate novel therapeutic approaches.

Haplotype-Assisted Noninvasive Prenatal Diagnosis of Genetic Diseases by Massively Parallel Sequencing of Maternal Plasma Cell-Free DNA.

Early prenatal diagnosis of genetic diseases allows for timely intervention or prevention of  the diseases in newborns. Conventional prenatal diagnosis of most genetic diseases relies on testing fetal DNA obtained by invasive procedures such as amniocentesis or chorionic villus sampling, which are associated with small risks of fetal loss. Maternal circulating blood contains cell-free DNA (cfDNA) from the fetal genome and can thus be used to noninvasively detect fetal genetic diseases such as chromosomal abnormalities, copy number variants, and single gene diseases. However, due to the presence of a high level of maternal cfDNA in the maternal blood stream, a relative haplotype dosage (RHDO) analysis is required to detect the mutant loci in the fetal genome when performing noninvasive prenatal diagnosis (NIPD) by massively parallel sequencing (MPS) of cfDNA. In this chapter, we describe a protocol utilizing the RHDO strategy for NIPD of any gene of interest associating with single gene diseases.

Water-soluble polythiophene-based colorimetry for the quick and accurate detection of SARS-CoV-2 RNA.

The SARS-CoV-2-related Corona Virus Disease 2019 (COVID-19) epidemic has had a significant negative impact on society and endangered global health. To quickly stop and constrain the pandemic, a SARS-CoV-2 detection technology that is sensitive, quick and reasonably priced is urgently required. The widely used reverse-transcription polymerase chain reaction (RT-PCR) requires complex equipment and a fair amount of time. Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) exhibits significant advantage for early detection of COVID-19 without the requirement for expensive equipment by amplifying a little amount of RNA to a detectable level at isothermal condition. Here, a water-soluble polythiophene-based colorimetric method by combining with RT-LAMP is established for fast and sensitive detection of SARS-CoV-2 RNA. The proposed assay has benefits for the quick detection of SARS-CoV-2 RNA at concentrations as low as 10 aM, or 6 copies/µL.

Double blocking gap-filling-ligation coupled with cascade isothermal amplification for ultrasensitive quantification of N6-methyladenosine.

We developed a method for quantifying N6-methyladenosine at one-nucleotide resolution based on double blocking gap-filling-ligation and cascade isothermal amplification. This proposed method can detect as low as 1 fM target RNA, achieving selectivity up to approximately 100-fold between m6A and A, and has been successfully applied to the analysis of m6A at specific sites in cell samples.

Multiple thermocycles followed by LAMP with only two primers for ultrasensitive colorimetric viral RNA testing and tracking at single-base resolution.

Rapid, accurate and high throughput measurement of infectious viruses is an urgent need to prevent viral transmission. Loop-mediated isothermal amplification (LAMP) is an attractive isothermal amplification method for nucleic acid detection, especially for point-of-care (POC) testing, but it needs at least four primers and its sensitivity is also limited when integrating with visual detection methods. Herein, by designing only two primers to precisely recognize the four regions of the target, we developed a multiple thermocycles-based LAMP method (MTC-LAMP) for sensitive and specific testing and tracking of viral RNA. We also introduced a novel SYBR Green I (SG)-assisted stable colorimetric assay induced by the amplification products through the charge neutralization effect of positively charged SG toward gold nanoparticles (AuNPs). The ultralow nonspecific background of the double exponential amplification improved the detection sensitivity to near single-molecule level (1 aM, 3 copies in 5 µL solution), which was higher than RT-PCR and RT-LAMP. After adding AuNPs, a significant color difference between target and blank was immediately observed by naked eye. By introducing a peptide nucleic acid (PNA) clamp into our colorimetric MTC-LAMP assay, the specific distinguish of virus variants at single-base resolution was observed without the requirement of any equipment. This assay shows great potential for large-scale screening and tracking of the threatening viruses with ultrahigh sensitivity and pronounced colorimetric output, which is of great importance for pandemic control.