Quantitative haplotype-resolved analysis of mitochondrial DNA heteroplasmy in Human single oocytes, blastoids, and pluripotent stem cells.

Maternal mitochondria are the sole source of mtDNA for every cell of the offspring. Heteroplasmic mtDNA mutations inherited from the oocyte are a common cause of metabolic diseases and associated with late-onset diseases. However, the origin and dynamics of mtDNA heteroplasmy remain unclear. We used our individual Mitochondrial Genome sequencing (iMiGseq) technology to study mtDNA heterogeneity, quantitate single nucleotide variants (SNVs) and large structural variants (SVs), track heteroplasmy dynamics, and analyze genetic linkage between variants at the individual mtDNA molecule level in single oocytes and human blastoids. Our study presented the first single-mtDNA analysis of the comprehensive heteroplasmy landscape in single human oocytes. Unappreciated levels of rare heteroplasmic variants well below the detection limit of conventional methods were identified in healthy human oocytes, of which many are reported to be deleterious and associated with mitochondrial disease and cancer. Quantitative genetic linkage analysis revealed dramatic shifts of variant frequency and clonal expansions of large SVs during oogenesis in single-donor oocytes. iMiGseq of a single human blastoid suggested stable heteroplasmy levels during early lineage differentiation of naïve pluripotent stem cells. Therefore, our data provided new insights of mtDNA genetics and laid a foundation for understanding mtDNA heteroplasmy at early stages of life.

Single-cell individual full-length mtDNA sequencing by iMiGseq uncovers unexpected heteroplasmy shifts in mtDNA editing.

The ontogeny and dynamics of mtDNA heteroplasmy remain unclear due to limitations of current mtDNA sequencing methods. We developed individual Mitochondrial Genome sequencing (iMiGseq) of full-length mtDNA for ultra-sensitive variant detection, complete haplotyping, and unbiased evaluation of heteroplasmy levels, all at the individual mtDNA molecule level. iMiGseq uncovered unappreciated levels of heteroplasmic variants in single cells well below the conventional NGS detection limit and provided accurate quantitation of heteroplasmy level. iMiGseq resolved the complete haplotype of individual mtDNA in single oocytes and revealed genetic linkage of de novo mutations. iMiGseq detected sequential acquisition of detrimental mutations, including large deletions, in defective mtDNA in NARP/Leigh syndrome patient-derived induced pluripotent stem cells. iMiGseq identified unintended heteroplasmy shifts in mitoTALEN editing, while showing no appreciable level of unintended mutations in DdCBE-mediated mtDNA base editing. Therefore, iMiGseq could not only help elucidate the mitochondrial etiology of diseases, but also evaluate the safety of various mtDNA editing strategies.

Modulation of the microhomology-mediated end joining pathway suppresses large deletions and enhances homology-directed repair following CRISPR-Cas9-induced DNA breaks.

BACKGROUND: CRISPR-Cas9 genome editing often induces unintended, large genomic rearrangements, posing potential safety risks. However, there are no methods for mitigating these risks. RESULTS: Using long-read individual-molecule sequencing (IDMseq), we found the microhomology-mediated end joining (MMEJ) DNA repair pathway plays a predominant role in Cas9-induced large deletions (LDs). We targeted MMEJ-associated genes genetically and/or pharmacologically and analyzed Cas9-induced LDs at multiple gene loci using flow cytometry and long-read sequencing. Reducing POLQ levels or activity significantly decreases LDs, while depleting or overexpressing RPA increases or reduces LD frequency, respectively. Interestingly, small-molecule inhibition of POLQ and delivery of recombinant RPA proteins also dramatically promote homology-directed repair (HDR) at multiple disease-relevant gene loci in human pluripotent stem cells and hematopoietic progenitor cells. CONCLUSIONS: Our findings reveal the contrasting roles of RPA and POLQ in Cas9-induced LD and HDR, suggesting new strategies for safer and more precise genome editing.

DeepSimulator1.5: a more powerful, quicker and lighter simulator for Nanopore sequencing.

MOTIVATION: Nanopore sequencing is one of the leading third-generation sequencing technologies. A number of computational tools have been developed to facilitate the processing and analysis of the Nanopore data. Previously, we have developed DeepSimulator1.0 (DS1.0), which is the first simulator for Nanopore sequencing to produce both the raw electrical signals and the reads. However, although DS1.0 can produce high-quality reads, for some sequences, the divergence between the simulated raw signals and the real signals can be large. Furthermore, the Nanopore sequencing technology has evolved greatly since DS1.0 was released. It is thus necessary to update DS1.0 to accommodate those changes. RESULTS: We propose DeepSimulator1.5 (DS1.5), all three modules of which have been updated substantially from DS1.0. As for the sequence generator, we updated the sample read length distribution to reflect the newest real reads' features. In terms of the signal generator, which is the core of DeepSimulator, we added one more pore model, the context-independent pore model, which is much faster than the previous context-dependent one. Furthermore, to make the generated signals more similar to the real ones, we added a low-pass filter to post-process the pore model signals. Regarding the basecaller, we added the support for the newest official basecaller, Guppy, which can support both GPU and CPU. In addition, multiple optimizations, related to multiprocessing control, memory and storage management, have been implemented to make DS1.5 a much more amenable and lighter simulator than DS1.0. AVAILABILITY AND IMPLEMENTATION: The main program and the data are available at https://github.com/lykaust15/DeepSimulator. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

DeepSimulator: a deep simulator for Nanopore sequencing.

Motivation: Oxford Nanopore sequencing is a rapidly developed sequencing technology in recent years. To keep pace with the explosion of the downstream data analytical tools, a versatile Nanopore sequencing simulator is needed to complement the experimental data as well as to benchmark those newly developed tools. However, all the currently available simulators are based on simple statistics of the produced reads, which have difficulty in capturing the complex nature of the Nanopore sequencing procedure, the main task of which is the generation of raw electrical current signals. Results: Here we propose a deep learning based simulator, DeepSimulator, to mimic the entire pipeline of Nanopore sequencing. Starting from a given reference genome or assembled contigs, we simulate the electrical current signals by a context-dependent deep learning model, followed by a base-calling procedure to yield simulated reads. This workflow mimics the sequencing procedure more naturally. The thorough experiments performed across four species show that the signals generated by our context-dependent model are more similar to the experimentally obtained signals than the ones generated by the official context-independent pore model. In terms of the simulated reads, we provide a parameter interface to users so that they can obtain the reads with different accuracies ranging from 83 to 97%. The reads generated by the default parameter have almost the same properties as the real data. Two case studies demonstrate the application of DeepSimulator to benefit the development of tools in de novo assembly and in low coverage SNP detection. Availability and implementation: The software can be accessed freely at: https://github.com/lykaust15/DeepSimulator. Supplementary information: Supplementary data are available at Bioinformatics online.

Pre-Treatment with Melatonin Enhances Therapeutic Efficacy of Cardiac Progenitor Cells for Myocardial Infarction.

BACKGROUND/AIMS: Melatonin possesses many biological activities such as antioxidant and anti-aging. Cardiac progenitor cells (CPCs) have emerged as a promising therapeutic strategy for myocardial infarction (MI). However, the low survival of transplanted CPCs in infarcted myocardium limits the successful use in treating MI. In the present study, we aimed to investigate if melatonin protects against oxidative stress-induced CPCs damage and enhances its therapeutic efficacy for MI. METHODS: TUNEL assay and EdU assay were used to detect the effects of melatonin and miR-98 on H2O2-induced apoptosis and proliferation. MI model was used to evaluate the potential cardioprotective effects of melatonin and miR-98. RESULTS: Melatonin attenuated H2O2-induced the proliferation reduction and apoptosis of c-kit+ CPCs in vitro, and CPCs which pretreated with melatonin significantly improved the functions of post-infarct hearts compared with CPCs alone in vivo. Melatonin was capable to inhibit the increase of miR-98 level by H2O2 in CPCs. The proliferation reduction and apoptosis of CPCs induced by H2O2 was aggravated by miR-98. In vivo, transplantation of CPCs with miR-98 silencing caused the more significant improvement of cardiac functions in MI than CPCs. MiR-98 targets at the signal transducer and activator of the transcription 3 (STAT3), and thus aggravated H2O2-induced the reduction of Bcl-2 protein. CONCLUSIONS: Pre-treatment with melatonin protects c-kit+ CPCs against oxidative stress-induced damage via downregulation of miR-98 and thereby increasing STAT3, representing a potentially new strategy to improve CPC-based therapy for MI.

Attenuation of Low Ambient Temperature-Induced Myocardial Hypertrophy by Atorvastatin via Promoting Bcl-2 Expression.

BACKGROUND/AIMS: It is well documented that myocardial hypertrophy is associated with low ambient temperature. Atorvastatin (Atv) has been shown to protect against atherosclerosis, cardiac fibrosis, ischemia/reperfusion injury, etc. In this study, we aim to determine whether atorvastatin is effective in the treatment of myocardial hypertrophy induced by cold exposure and to shed light on underlying mechanism. METHODS: The mice aged 4-week were randomized to Control (Ctl) group (raised at room temperature), Cold group (raised at 3-5ºC) and Atv treatment group (raised at 3-5ºC followed by 10mg/kg/day Atv infusion). Echocardiography (ECG), HE, TUNEL and Masson's trichrome staining, and Transmission electronic microscopy were performed to analyze cardiac function, myocardial hypertrophy, cardiac fibrosis, apoptosis and cardiomyocyte ultrastructure, respectively. Western blot was carried out to determine the involvement of MAPK and apoptosis pathways. RESULTS: Exposure of mice to low temperature induced myocardial hypertrophic growth characterized by the elevation of heart/body weight index and heart weight /tibia length index, compared with control mice. Atv treatment attenuated cardiac hypertrophy induced by cold exposure; Atv also attenuated the increase of cross-sectional area of cardiomyocytes and cardiac collagen content fraction in mice exposed to cold. ECG showed that the decline of cardiac functions including the elevated left ventricular systolic/diastolic internal dimension (LVIDs/d) and fractional shortening (FS) in mice with cold exposure was also inhibited by Atv treatment. Transmission electronic microscopy uncovered that Atv attenuated mitochondrial injury induced by cold exposure in mice. In addition, systolic blood pressure was gradually increased in mice exposed to cold temperature, and Atv treatment significantly inhibited the elevation of blood pressure in cold-treated mice. Mechanistically, mitogen-activated protein kinase (MAPK) signal was not altered in mice exposed to cold, and Atv did not affect MAPK signal in cold-treated mice. But Atv mitigated the reduction of Bcl-2/Bax level in heart of cold-treated mice. CONCLUSION: Atv attenuated myocardial hypertrophy induced by cold exposure through inhibiting the downregulation of Bcl-2 in heart. It may provide a novel strategy for low temperature-induced myocardial hypertrophy treatment.

Astragalus Polysaccharide Attenuated Iron Overload-Induced Dysfunction of Mesenchymal Stem Cells via Suppressing Mitochondrial ROS.

BACKGROUND/AIMS: Bone marrow-derived mesenchymal stem cells (BMSCs) have the ability to differentiate into multilineage cells such as osteoblasts, chondrocytes, and cardiomyocytes. Dysfunction of BMSCs in response to pathological stimuli participates in the development of diseases such as osteoporosis. Astragalus polysaccharide (APS) is a major active ingredient of Astragalus membranaceus, a commonly used anti-aging herb in traditional Chinese medicine. The aim of this study was to investigate whether APS protects against iron overload-induced dysfunction of BMSCs and its underlying mechanisms. METHODS: BMSCs were exposed to ferric ammonium citrate (FAC) with or without different concentrations of APS. The viability and proliferation of BMSCs were assessed by CCK-8 assay and EdU staining. Cell apoptosis, senescence and pluripotency were examined utilizing TUNEL staining, ß-galactosidase staining and qRT-PCR respectively. The reactive oxygen species (ROS) level was assessed in BMSCs with a DCFH-DA probe and MitoSOX Red staining. RESULTS: Firstly, we found that iron overload induced by FAC markedly reduced the viability and proliferation of BMSCs, but treatment with APS at 10, 30 and 100 µg/mL was able to counter the reduction of cell proliferation. Furthermore, exposure to FAC led to apoptosis and senescence in BMSCs, which were partially attenuated by APS. The pluripotent genes Nanog, Sox2 and Oct4 were shown to be downregulated in BMSCs after FAC treatment, however APS inhibited the reduction of Nanog, Sox2 and Oct4 expression. Further study uncovered that APS treatment abrogated the increase of intracellular and mitochondrial ROS level in FAC-treated BMSCs. CONCLUSION: Treatment of BMSCs with APS to impede mitochondrial ROS accumulation can remarkably inhibit apoptosis, senescence, and the reduction of proliferation and pluripotency of BMSCs caused by FAC-induced iron overload.

Long noncoding RNA H19 mediates melatonin inhibition of premature senescence of c-kit(+) cardiac progenitor cells by promoting miR-675.

Melatonin, a hormone secreted by the pineal gland, possesses multiple biological activities such as antitumor, antioxidant, and anti-ischemia. C-kit(+) cardiac progenitor cells (CPCs) have emerged as a promising tool for the treatment of heart diseases. However, the senescence of CPCs due to pathological stimuli leads to the decline of CPCs' functions and regenerative potential. This study was conducted to demonstrate whether melatonin antagonizes the senescence of CPCs in response to oxidative stress. Here, we found that the melatonin treatment markedly inhibited the senescent characteristics of CPCs after exposed to sublethal concentration of H2 O2 , including the increase in senescence-associated ß-galactosidase (SA-ß-gal)-positive CPCs, senescence-associated heterochromatin loci (SAHF), secretory IL-6 level, and the upregulation of p53 and p21 proteins. Senescence-associated proliferation reduction was also attenuated by melatonin in CPCs. Luzindole, the melatonin membrane receptor blocker, may block the melatonin-mediated suppression of premature senescence in CPCs. Interestingly, we found that long noncoding RNA H19 and its derived miR-675 were downregulated by H2 O2 in CPCs, but melatonin treatment could counter this alteration. Furthermore, knockdown of H19 or miR-675 blocked antisenescence actions of melatonin on H2 O2 -treated CPCs. It was further verified that H19-derived miR-675 targeted at the 3'UTR of USP10, which resulted in the downregulation of p53 and p21 proteins. In summary, melatonin antagonized premature senescence of CPCs via H19/miR-675/USP10 pathway, which provides new insights into pharmacological actions and potential applications of melatonin on the senescence of CPCs.

The use of chlorogenic acid and its analogues as inhibitors: an investigation of the inhibition of sortase A of Staphylococcus aureus using molecular docking and dynamic simulation.

OBJECTIVES: To use molecular docking and dynamic simulation to investigate the inhibitory action of chlorogenic acid (CHA) and its analogues against sortase A of Staphylococcus aureus. RESULTS: Five novel, natural inhibitors with different activities were discovered for sortase A (SrtA). The inhibition mechanism of the novel inhibitors was consistent with the mechanism of CHA, which was reported previously by Wang et al. (Front Microbiol 6:1031, 2015). Based on structure-activity relationship analysis, the hydroxyl moiety (C1) of the inhibitors is critical in the catalytic region of SrtA, which could be confirmed by the calculation of the binding free energy between SrtA and the inhibitors. CONCLUSIONS: The mechanism obtained by molecular dynamics simulation is thus useful for the development of novel, selective SrtA inhibitors.

Acacetin Protects Mice from Staphylococcus aureus Bloodstream Infection by Inhibiting the Activity of Sortase A.

Staphylococcus aureus (S. aureus) is a major cause of infection in hospitals and communities. Widespread dissemination of multi-drug resistant S. aureus is a serious threat to the health of humans and animals. An anti-virulence strategy has been widely considered as an alternative therapeutic approach. Inhibitors of virulence factors are able to treat S. aureus infections without influencing the growth or viability of bacteria and rarely lead to bacterial resistance. Sortase A (SrtA) is a membrane-associated cysteine transpeptidase that catalyzes up to 25 surface proteins that covalently bind to cell wall peptidoglycans. In S. aureus, most of these surface proteins have been identified as important virulence factors that are vital in bacterial pathogenesis. In the present study, we show that acacetin, a natural flavonoid compound, inhibits the activity of SrtA in S. aureus (IC50 = 36.46 ± 4.69 µg/mL, 128 µM) which affects the assembly of protein A (SpA) to cell walls and reduces the binding of S. aureus to fibrinogen (Fg). The mechanism of the interaction between acacetin and SrtA were preliminarily discussed using molecular dynamics simulations. The results suggested that acacetin adopted a compact conformation binding at the pocket of the SrtA via residues Arg-139 and Lys-140. By performing an animal infection model, we demonstrated that acacetin was able to protect mice from renal abscess formation induced by S. aureus and significantly increased survival rates. Taken together, these findings suggest that acacetin may be a promising candidate for the development of anti-S. aureus drugs.

Resveratrol Attenuated Low Ambient Temperature-Induced Myocardial Hypertrophy via Inhibiting Cardiomyocyte Apoptosis.

BACKGROUND/AIMS: Low ambient temperature is an important risk factor for cardiovascular diseases, and has been shown to lead to cardiac hypertrophy. In this study, we aim to investigate if Resveratrol may inhibit cold exposure-induced cardiac hypertrophy in mice, and if so to clarify its molecular mechanism. METHODS: Adult male mice were randomly assigned to Control group (kept at room temperature), Cold group (kept at low air temperature range from 3°C to 5°C) and Resveratrol treatment group (100mg/kg/day) for eight weeks. HE staining, Masson staining and Transmission electron microscopy were employed to detect cardiac structure, fibrosis and myocardial ultrastructure, respectively. Echocardiogram was used to measure myocardial functions. Western blot was used to detect the expression of MAPK pathway and apoptotic proteins. TUENL assay was performed to evaluate cardiomyocyte apoptosis. qRT-PCR was employed to measure the mRNA level. RESULTS: Cold-treated mice showed a higher heart/body weight ratio and heart weight/tibia length ratio compared with control mice, and Resveratrol treatment may suppress these changes in cold-treated mice. Myocardial cross-section area and cardiac collagen volume were larger in cold group than control group, which also can be attenuated by Resveratrol treatment. Also, Resveratrol improved the ultrastructure damage of myocardium such as myofibril disarray in cold group. Echocardiogram measurement showed that EF and FS values in cold group declined apparently as compared to control group, and Resveratrol may improve the reduction of heart functions. The expression of p-JNK, p-p38 and p-ERK relative to total JNK, p38 and ERK in cold group was not altered in cold group and Resveratrol group as compared to control group. Cold-treated mouse hearts also showed the upregulation of hypertrophy-related miRNA-miR-328 but not miR-23a, and Resveratrol treatment can inhibit the increase of miR-328. Finally, Resveratrol treatment also may suppress apoptosis of myocardium in cold-treated mouse hearts via inhibiting Bax and caspase-3 activation. CONCLUSION: In summary, low ambient temperature can cause enlarged heart, ultrastructure damage of myocardium and weakened functions, and Resveratrol treatment effectively suppressed these changes at least partially via inhibiting cardiomyocyte apoptosis.

Quercitrin, an inhibitor of Sortase A, interferes with the adhesion of Staphylococcal aureus.

Sortase A (SrtA) is a cysteine transpeptidase of most Gram-positive bacteria that is responsible for the anchorage of many surface protein virulence factors to the cell wall layer. SrtA mutants are unable to display surface proteins and are defective in the establishment of infections without affecting microbial viability. In this study, we report that quercitrin (QEN), a natural compound that does not affect Staphylococcus aureus growth, can inhibit the catalytic activity of SrtA in fibrinogen (Fg) cell-clumping and immobilized fibronectin (Fn) adhesion assays. Molecular dynamics simulations and mutagenesis assays suggest that QEN binds to the binding sites of the SrtA G167A and V193A mutants. These findings indicate that QEN is a potential lead compound for the development of new anti-virulence agents against S. aureus infections.

NanoRanger enables rapid single-base-pair resolution of genomic disorders.

BACKGROUND: Delineating base-resolution breakpoints of complex rearrangements is crucial for an accurate clinical understanding of pathogenic variants and for carrier screening within family networks or the broader population. However, despite advances in genetic testing using short-read sequencing (SRS), this task remains costly and challenging. METHODS: This study addresses the challenges of resolving missing disease-causing breakpoints in complex genomic disorders with suspected homozygous rearrangements by employing multiple long-read sequencing (LRS) strategies, including a novel and efficient strategy named nanopore-based rapid acquisition of neighboring genomic regions (NanoRanger). NanoRanger does not require large amounts of ultrahigh-molecular-weight DNA and stands out for its ease of use and rapid acquisition of large genomic regions of interest with deep coverage. FINDINGS: We describe a cohort of 16 familial cases, each harboring homozygous rearrangements that defied breakpoint determination by SRS and optical genome mapping (OGM). NanoRanger identified the breakpoints with single-base-pair resolution, enabling accurate determination of the carrier status of unaffected family members as well as the founder nature of these genomic lesions and their frequency in the local population. The resolved breakpoints revealed that repetitive DNA, gene regulatory elements, and transcription activity contribute to genome instability in these novel recessive rearrangements. CONCLUSIONS: Our data suggest that NanoRanger greatly improves the success rate of resolving base-resolution breakpoints of complex genomic disorders and expands access to LRS for the benefit of patients with Mendelian disorders. FUNDING: M.L. is supported by KAUST Baseline Award no. BAS/1/1080-01-01 and KAUST Research Translation Fund Award no. REI/1/4742-01.

Quantification of Genetic Heterogeneity Using Long-Read Targeted Individual DNA Molecule Sequencing.

Understanding genetic heterogeneity is of paramount importance in unraveling the intricate functioning of biological systems, as it contributes to the diversity of phenotypes of gene-environment interactions. We have developed a method termed targeted Individual DNA Molecule Sequencing (IDMseq) to accurately quantify genetic heterogeneity within cell populations, even those with rare variants present at low frequencies. IDMseq ensures that each original DNA molecule is distinctively represented by one unique molecule identifier (UMI) group, preventing false UMI groups and enabling precise quantification of allele frequency within the original population. IDMseq is a versatile sequencing technique that combines error correction and long-read sequencing, enabling sensitive detection of various genetic variants, including single nucleotide variants and large structural variants in both basic and clinical research settings. This protocol provides a comprehensive, step-by-step guide to preparing samples and performing IDMseq to determine genetic variations. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: UMI labeling and amplification of DNA Support Protocol 1: AMPure XP beads cleanup Support Protocol 2: Suggested data analysis pipeline.

An open-source, automated, and cost-effective platform for COVID-19 diagnosis and rapid portable genomic surveillance using nanopore sequencing.

The COVID-19 pandemic, caused by SARS-CoV-2, has emphasized the necessity for scalable diagnostic workflows using locally produced reagents and basic laboratory equipment with minimal dependence on global supply chains. We introduce an open-source automated platform for high-throughput RNA extraction and pathogen diagnosis, which uses reagents almost entirely produced in-house. This platform integrates our methods for self-manufacturing magnetic nanoparticles and qRT-PCR reagents-both of which have received regulatory approval for clinical use-with an in-house, open-source robotic extraction protocol. It also incorporates our "Nanopore Sequencing of Isothermal Rapid Viral Amplification for Near Real-time Analysis" (NIRVANA) technology, designed for tracking SARS-CoV-2 mutations and variants. The platform exhibits high reproducibility and consistency without cross-contamination, and its limit of detection, sensitivity, and specificity are comparable to commercial assays. Automated NIRVANA effectively identifies circulating SARS-CoV-2 variants. Our in-house, cost-effective reagents, automated diagnostic workflows, and portable genomic surveillance strategies provide a scalable and rapid solution for COVID-19 diagnosis and variant tracking, essential for current and future pandemic responses.

NIRVANA for Simultaneous Detection and Mutation Surveillance of SARS-CoV-2 and Co-infections of Multiple Respiratory Viruses.

Detection and mutation surveillance of SARS-CoV-2 are crucial for combating the COVID-19 pandemic. Here we describe a lab-based method for multiplex isothermal amplification-based sequencing and real-time analysis of multiple viral genomes. It can simultaneously detect SARS-CoV-2, influenza A, human adenovirus, and human coronavirus and monitor mutations for up to 96 samples in real time. The method proved to be rapid and sensitive (limit of detection: 29 viral RNA copies/µL of extracted nucleic acid) in detecting SARS-CoV-2 in clinical samples. We expect it to offer a promising solution for rapid field-deployable detection and mutational surveillance of pandemic viruses.

Wiskott-Aldrich syndrome protein forms nuclear condensates and regulates alternative splicing.

The diverse functions of WASP, the deficiency of which causes Wiskott-Aldrich syndrome (WAS), remain poorly defined. We generated three isogenic WAS models using patient induced pluripotent stem cells and genome editing. These models recapitulated WAS phenotypes and revealed that WASP deficiency causes an upregulation of numerous RNA splicing factors and widespread altered splicing. Loss of WASP binding to splicing factor gene promoters frequently leads to aberrant epigenetic activation. WASP interacts with dozens of nuclear speckle constituents and constrains SRSF2 mobility. Using an optogenetic system, we showed that WASP forms phase-separated condensates that encompasses SRSF2, nascent RNA and active Pol II. The role of WASP in gene body condensates is corroborated by ChIPseq and RIPseq. Together our data reveal that WASP is a nexus regulator of RNA splicing that controls the transcription of splicing factors epigenetically and the dynamics of the splicing machinery through liquid-liquid phase separation.

Simultaneous detection and mutation surveillance of SARS-CoV-2 and multiple respiratory viruses by rapid field-deployable sequencing.

BACKGROUND: Strategies for monitoring the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are crucial for combating the pandemic. Detection and mutation surveillance of SARS-CoV-2 and other respiratory viruses require separate and complex workflows that rely on highly specialized facilities, personnel, and reagents. To date, no method can rapidly diagnose multiple viral infections and determine variants in a high-throughput manner. METHODS: We describe a method for multiplex isothermal amplification-based sequencing and real-time analysis of multiple viral genomes, termed nanopore sequencing of isothermal rapid viral amplification for near real-time analysis (NIRVANA). It can simultaneously detect SARS-CoV-2, influenza A, human adenovirus, and human coronavirus and monitor mutations for up to 96 samples in real time. FINDINGS: NIRVANA showed high sensitivity and specificity for SARS-CoV-2 in 70 clinical samples with a detection limit of 20 viral RNA copies per µL of extracted nucleic acid. It also detected the influenza A co-infection in two samples. The variant analysis results of SARS-CoV-2-positive samples mirror the epidemiology of coronavirus disease 2019 (COVID-19). Additionally, NIRVANA could simultaneously detect SARS-CoV-2 and pepper mild mottle virus (PMMoV) (an omnipresent virus and water-quality indicator) in municipal wastewater samples. CONCLUSIONS: NIRVANA provides high-confidence detection of both SARS-CoV-2 and other respiratory viruses and mutation surveillance of SARS-CoV-2 on the fly. We expect it to offer a promising solution for rapid field-deployable detection and mutational surveillance of pandemic viruses. FUNDING: M.L. is supported by KAUST Office of Sponsored Research (BAS/1/1080-01). This work is supported by KAUST Competitive Research Grant (URF/1/3412-01-01; M.L. and J.C.I.B.) and Universidad Catolica San Antonio de Murcia (J.C.I.B.). A.M.H. is supported by Saudi Ministry of Education (project 436).

Long-read individual-molecule sequencing reveals CRISPR-induced genetic heterogeneity in human ESCs.

Quantifying the genetic heterogeneity of a cell population is essential to understanding of biological systems. We develop a universal method to label individual DNA molecules for single-base-resolution haplotype-resolved quantitative characterization of diverse types of rare variants, with frequency as low as 4 × 10-5, using both short- or long-read sequencing platforms. It provides the first quantitative evidence of persistent nonrandom large structural variants and an increase in single-nucleotide variants at the on-target locus following repair of double-strand breaks induced by CRISPR-Cas9 in human embryonic stem cells.