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Embryonic stem (ES) cells retain the ability to undergo lineage-specific differentiation that can eventually give rise to different cell types that constitute an organism. Although stem cell specific biological networks of transcription factors and epigenetic modifiers are well established, how the ES cell specific transcriptional and alternative splicing (AS) machinery regulate their expression has not been sufficiently explored. In this study, we show that the lncRNA associated protein TOBF1 regulates the co-transcriptional alternative splicing of transcripts necessary for maintaining stem cell identity in mouse ES cells. Overlaying information derived from TOBF1 chromatin occupancy, the distribution of its pluripotency-associated OCT-SOX binding motifs, and transcripts undergoing differential expression and alternative splicing upon its disruption unmasked local nuclear territories where these distinct events converge, ultimately leading to the maintenance of mouse ES cell identity.

MALAT1, an abundant lncRNA specifically localized to nuclear speckles, regulates alternative-splicing (AS). The molecular basis of its role in AS remains poorly understood. Here, we report three conserved, thermodynamically stable, parallel RNA-G-quadruplexes (rG4s) present in the 3′ region of MALAT1 which regulates this function. Using rG4 domain-specific RNA-pull-down followed by mass-spectrometry, RNA-immuno-precipitation, and imaging, we demonstrate the rG4 dependent localization of Nucleolin (NCL) and Nucleophosmin (NPM) to nuclear speckles. Specific G-to-A mutations that abolish rG4 structures, result in the localization loss of both the proteins from speckles. Functionally, disruption of rG4 in MALAT1 phenocopies NCL knockdown resulting in altered pre-mRNA splicing of endogenous genes. These results reveal a central role of rG4s within the 3′ region of MALAT1 orchestrating AS.

Nucleic acid detection and variant calling through CRISPR-based diagnostics (CRISPRDx) has facilitated clinical decision-making, particularly during the COVID-19 pandemic. This has been further accelerated through the discovery of newer and engineered CRISPR effectors, expanding the portfolio of such diagnostic applications to a wide variety of pathogenic and non-pathogenic conditions. However, each diagnostic CRISPR pipeline requires customized detection schemes originating from fundamental principles of the Cas protein used, its guide RNA (gRNA) design parameters, and the assay readout. This is particularly relevant for variant detection, an attractive low-cost alternative to sequencing-based approaches for which no in silico pipeline for the ready-to-use design of CRISPR-based diagnostics currently exists. In this manuscript, we fill this lacuna using a unified webserver CriSNPr (CRISPR based SNP recognition), which provides the user the opportunity to de-novo design gRNAs based on six CRISPRDx proteins of choice (Fn/enFnCas9, LwCas13a, LbCas12a, AaCas12b, and Cas14a) and query for ready-to-use oligonucleotide sequences for validation on relevant samples. In addition, we provide a database of curated pre-designed gRNAs and target/off-target for all human and SARS-CoV-2 variants reported so far. CriSNPr has been validated on multiple Cas proteins and highlights its broad and immediate scope of utilization across multiple detection platforms. CriSNPr is available at URL http://crisnpr.igib.res.in/.

The triple-helix structure at the 3' end of metastasis-associated lung adenocarcinoma transcript 1 (

The characteristics of immune memory established in response to inactivated SARS-CoV-2 vaccines remains unclear. We determined the magnitude, quality and persistence of cellular and humoral memory responses up to 6 months after vaccination with BBV152/Covaxin. Here, we show that the quantity of vaccine-induced spike- and nucleoprotein-antibodies is comparable to that following natural infection and the antibodies are detectable up to 6 months. The RBD-specific antibodies decline in the range of 3 to 10-fold against the SARS-CoV-2 variants in the order of alpha (B.1.1.7) > delta (B.1.617.2) > beta (B.1.351), with no observed impact of gamma (P.1) and kappa (B.1.617.1) variant. We found that the vaccine induces memory B cells, similar to natural infection, which are impacted by virus variants in the same order as antibodies. The vaccine further induced antigen-specific functionally potent multi-cytokine expressing CD4+ T cells in ∼85% of the subjects, targeting spike and nucleoprotein of SARS-CoV-2. Marginal ∼1.3 fold-reduction was observed in vaccine-induced CD4+ T cells against the beta variant, with no significant impact of the alpha and the delta variants. The antigen-specific CD4+ T cells were populated in the central memory compartment and persisted up to 6 months of vaccination. Importantly the vaccine generated Tfh cells that are endowed with B cell help potential, similar to the Tfh cells induced after natural infection. Altogether, these findings establish that the inactivated virus vaccine BBV152 induces robust immune memory to SARS-CoV-2 and variants of concern, which persist for at least 6 months after vaccination. This study provides insight into the attributes of BBV152-elicited immune memory, and has implication for future vaccine development, guidance for use of inactivated virus vaccine, and booster immunization.

The clinical success of CRISPR therapies is dependent on the safety and efficacy ofCas proteins. The Cas9 from Francisella novicida (FnCas9) has negligible affinity formismatched substrates enabling it to discriminate off-targets in DNA with very highprecision even at the level of binding. However, its cellular targeting efficiency is low,limiting its use in therapeutic applications. Here, we rationally engineer the protein todevelop engineered(enFnCas9) variants with enhanced activity and expand its cellularediting activity to genomic loci previously inaccessible. Notably, some of the variantsrelease the protospacer adjacent motif (PAM) constraint from NGG to NGR/NRGmaking them rank just below SpCas9-RY and SpCas9-NG in their accessibility acrosshuman genomic sites. The enFnCas9 proteins, similar to Cas12a and Cas12f, harborhigh intrinsic specificity and can diagnose single nucleotide variants accurately.Importantly, they provide superior outcomes in terms of editing efficiency, knock-inrates, and off-target specificity over other engineered high-fidelity versions of SpCas9(SpCas9-HF1 and eSpCas9). Broad targeting range coupled with remarkablespecificity of DNA interrogation underscores the utility of these variants for safe andefficient therapeutic gene correction across multiple cell lines and target loci.

Introduction: Breast cancer is the most frequently diagnosed cancer globally and is one of the most important contributors to cancer-related deaths. Earlier diagnosis is known to reduce mortality, and better biomarkers are needed. MiRNA clusters often co-express and target mRNAs in a coordinated fashion, perturbing entire pathways, they thus merit further exploration for diagnostic or prognostic use. MiR-379/656, at chromosome 14q32, is the second largest miRNA cluster in the human genome and implicated in various malignancies including glioblastoma, melanoma, gastrointestinal tumors and ovarian cancer highlighting its potential importance. In this study, we focus on the diagnostic and prognostic potentials of MiR-379/656 in breast cancer and its molecular subtypes. Materials and Methods: We analyzed miRNA and mRNA next generation sequencing data from 903 primary tumors and 90 normal controls (source: The Cancer Genome Atlas). The differential expression profile between tumor and normal was analyzed using DeSEQ2. Penalized logistic regression modelling (lasso regression) was used to assess the predictive potential of MiR-379/656 expression for tumor and normal samples. The association between MiR-379/656 expression and overall patient survival was studied using Cox Proportional-Hazard Model. The target mRNAs (validated) of MiR-379/656 were annotated via pathway enrichment analysis to understand the biological significance of the cluster in breast cancer. Results: The differential expression analysis for 1390 miRNAs (miRnome) revealed 310 upregulated (22.3%) and 176 downregulated (12.66%) miRNAs in breast cancer patients compared with controls. For MiR-379/656, 32 miRNAs (32/42, 76%) were downregulated. The MiR-379/656 cluster was found to be the most differentially expressed cluster in the human genome (p &lt, 10−30). The Basal and Luminal B subtypes showed at least 83% (35/42) of the miRNAs to be downregulated. The binomial model prioritized 15 miRNAs, which distinguished breast cancer patients from controls with 99.15 ± 0.58% sensitivity and 77.78 ± 5.24% specificity. Overall, the Basal and Luminal B showed the most effective predictive power with respect to the 15 prioritized miRNAs at MiR-379/656 cluster. The decreased expression of MiR-379/656 was found to be associated with poorer clinical outcome in Basal and Luminal B subtypes, increasing tumor stage and tumor size/extent, and overall patient survival. Pathway enrichment for the validated targets of MiR-379/656 was significant for cancer-related pathways, especially DNA repair, transcriptional regulation by p53 and cell cycle checkpoints (adjusted p-value &lt, 0.05). Conclusions: Genome informatics analysis of high throughput data for MiR-379/656 cluster has shown that a subset of 15 miRNAs from MiR-379/656 cluster can be used for the diagnostic and prognostic purpose of breast cancer and its subtypes—especially in Basal and Luminal B.

SummaryThe B cell help function of CD4+T cells may serve as an immunologic correlate of protective adaptive immunity. The quantitative assessment of the B cell help potential of CD4+T cells is limited by the lack of suitable antigen-specific functional assays. Here, we describe a highly efficient antigen-specific T-B co-cultures for quantitative measurement of T-dependent B cell responses. UsingMycobacterium tuberculosisspecific setup, we show that early priming and activation of CD4+T cells is important for the mutualistic collaboration between antigen-specific T and B cells, which could be achieved by supplementing the co-cultures with autologous monocytes. We further show that monocyte-derived growth factors provide the impetus for productive T-B collaboration by conferring optimal survivability in the cultured cells. This study provides first evidence of C-type lectin domain family 11 member A (CLEC11A/SCGF) as an essential growth factor for B cell survival. Importantly, we demonstrate the successful translation of monocyte supplemented T-B co-cultures in qualitative assessment of SARS-CoV-2 specific memory CD4+T cells by quantifying several correlates of productive T-B cross-talk like plasma cell output, secreted antibody, antibody secreting cells and IL21 secreting T cells. Thus, the method described here can provides qualitative assessment of SARS-CoV-2 spike CD4+T cells after natural infection and can be applied to assess the B cell help function of memory CD4+T cells generated in response to COVID-19 vaccine.One sentence summaryQualitative assessment of antigen-specific CD4+T cells for T-dependent B cell responses.

The COVID-19 pandemic originating in the Wuhan province of China in late 2019 has impacted global health, causing increased mortality among elderly patients and individuals with comorbid conditions. During the passage of the virus through affected populations, it has undergone mutations, some of which have recently been linked with increased viral load and prognostic complexities. Several of these variants are point mutations that are difficult to diagnose using the gold standard quantitative real-time PCR (qRT-PCR) method and necessitates widespread sequencing which is expensive, has long turn-around times, and requires high viral load for calling mutations accurately. Here, we repurpose the high specificity of Francisella novicida Cas9 (FnCas9) to identify mismatches in the target for developing a lateral flow assay that can be successfully adapted for the simultaneous detection of SARS-CoV-2 infection as well as for detecting point mutations in the sequence of the virus obtained from patient samples. We report the detection of the S gene mutation N501Y (present across multiple variant lineages of SARS-CoV-2) within an hour using lateral flow paper strip chemistry. The results were corroborated using deep sequencing on multiple wild-type (n = 37) and mutant (n = 22) virus infected patient samples with a sensitivity of 87% and specificity of 97%. The design principle can be rapidly adapted for other mutations (as shown also for E484K and T716I) highlighting the advantages of quick optimization and roll-out of CRISPR diagnostics (CRISPRDx) for disease surveillance even beyond COVID-19. This study was funded by Council for Scientific and Industrial Research, India., eLife digest SARS-CoV-2, the virus responsible for COVID-19, has a genome made of RNA (a nucleic acid similar to DNA) that can mutate, potentially making the disease more transmissible, and more lethal. Most countries have monitored the rise of mutated strains using a technique called next generation sequencing (NGS), which is time-consuming, expensive and requires skilled personnel. Sometimes the mutations to the virus are so small that they can only be detected using NGS. Finding cheaper, simpler and faster SARS-CoV-2 tests that can reliably detect mutated forms of the virus is crucial for public health authorities to monitor and manage the spread of the virus. Lateral flow tests (the same technology used in many pregnancy tests) are typically cheap, fast and simple to use. Typically, lateral flow assay strips have a band of immobilised antibodies that bind to a specific protein (or antigen). If a sample contains antigen molecules, these will bind to the immobilised antibodies, causing a chemical reaction that changes the colour of the strip and giving a positive result. However, lateral flow tests that use antibodies cannot easily detect nucleic acids, such as DNA or RNA, let alone mutations in them. To overcome this limitation, lateral flow assays can be used to detect a protein called Cas9, which, in turn, is able to bind to nucleic acids with specific sequences. Small changes in the target sequence change how well Cas9 binds to it, meaning that, in theory, this approach could be used to detect small mutations in the SARS-CoV-2 virus. Kumar et al. made a lateral flow test that could detect a Cas9 protein that binds to a nucleic acid sequence found in a specific mutant strain of SARS-CoV-2. This Cas9 was highly sensitive to changes in its target sequence, so a small mutation in the target nucleic acid led to the protein binding less strongly, and the signal from the lateral flow test being lost. This meant that the lateral flow test designed by Kumar et al. could detect mutations in the SARS-CoV-2 virus at a fraction of the price of NGS approaches if used only for diagnosis. The lateral flow test was capable of detecting mutant viruses in patient samples too, generating a colour signal within an hour of a positive sample being run through the assay. The test developed by Kumar et al. could offer public health authorities a quick and cheap method to monitor the spread of mutant SARS-CoV-2 strains; as well as a way to determine vaccine efficacy against new strains.

Rapid detection of DNA/RNA pathogenic sequences or variants through point-of-care diagnostics is valuable for accelerated clinical prognosis, as witnessed during the recent COVID-19 outbreak. Traditional methods relying on qPCR or sequencing are tough to implement with limited resources, necessitating the development of accurate and robust alternative strategies. Here, we report FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that utilizes a direct Cas9 based enzymatic readout for detecting nucleobase and nucleotide sequences without trans-cleavage of reporter molecules. We also demonstrate that FELUDA is 100% accurate in detecting single nucleotide variants (SNVs), including heterozygous carriers, and present a simple web-tool JATAYU to aid end-users. FELUDA is semi-quantitative, can adapt to multiple signal detection platforms, and deploy for versatile applications such as molecular diagnosis during infectious disease outbreaks like COVID-19. Employing a lateral flow readout, FELUDA shows 100% sensitivity and 97% specificity across all ranges of viral loads in clinical samples within 1hr. In combination with RT-RPA and a smartphone application True Outcome Predicted via Strip Evaluation (TOPSE), we present a prototype for FELUDA for CoV-2 detection closer to home.

Understanding the causes of the diverse outcome of COVID-19 pandemic in different geographical locations is important for the worldwide vaccine implementation and pandemic control responses. We analyzed 42 unexposed healthy donors and 28 mild COVID-19 subjects up to 5 months from the recovery for SARS-CoV-2 specific immunological memory. Using HLA class II predicted peptide megapools, we identified SARS-CoV-2 cross-reactive CD4+ T cells in around 66% of the unexposed individuals. Moreover, we found detectable immune memory in mild COVID-19 patients several months after recovery in the crucial arms of protective adaptive immunity; CD4+ T cells and B cells, with a minimal contribution from CD8+ T cells. Interestingly, the persistent immune memory in COVID-19 patients is predominantly targeted towards the Spike glycoprotein of the SARS-CoV-2. This study provides the evidence of both high magnitude pre-existing and persistent immune memory in Indian population. By providing the knowledge on cellular immune responses to SARS-CoV-2, our work has implication for the development and implementation of vaccines against COVID-19.

The COVID-19 pandemic originating in the Wuhan province of China in late 2019 has impacted global health, causing increased mortality among elderly patients and individuals with comorbid conditions. During the passage of the virus through affected populations, it has undergone mutations- some of which have recently been linked with increased viral load and prognostic complexities. Interestingly, several of these variants are point mutations that are difficult to diagnose using the gold standard quantitative real-time PCR (qPCR) method. This necessitates widespread sequencing which is expensive, has long turn-around times, and requires high viral load for calling mutations accurately. In this study, we show that the high specificity of Francisella novicida Cas9 (FnCas9) to point mismatches can be successfully adapted for the simultaneous detection of SARS-CoV2 infection as well as for detecting point mutations in the sequence of the virus obtained from patient samples. We report the detection of the mutation N501Y (earlier shown to be present in the British N501Y.V1, South African N501Y.V2, and Brazilian N501Y.V3 variants of SARS-CoV2) within an hour using paper strip chemistry. The results were corroborated using deep sequencing. Our design principle can be rapidly adapted for other mutations, highlighting the advantages of quick optimization and roll-out of CRISPR diagnostics (CRISPRDx) for disease surveillance even beyond COVID-19.

The circadian clock regulates vital cellular processes by adjusting the physiology of the organism to daily changes in the environment. Rhythmic transcription of core Clock Genes (CGs) and their targets regulate these processes at the cellular level. Circadian clock disruption has been observed in people with neurodegenerative disorders like Alzheimer’s and Parkinson’s. Also, ablation of CGs during development has been shown to affect neurogenesis in both in vivo and in vitro models. Previous studies on the function of CGs in the brain have used knock-out models of a few CGs. However, a complete catalog of CGs in different cell types of the developing brain is not available and it is also tedious to obtain. Recent advancements in single-cell RNA sequencing (scRNA-seq) has revealed novel cell types and elusive dynamic cell states of the developing brain. In this study by using publicly available single-cell transcriptome datasets we systematically explored CGs-coexpressing networks (CGs-CNs) during embryonic and adult neurogenesis. Our meta-analysis reveals CGs-CNs in human embryonic radial glia, neurons and also in lesser studied non-neuronal cell types of the developing brain.

Rapid detection of pathogenic sequences or variants in DNA and RNA through a point-of-care diagnostic approach is valuable for accelerated clinical prognosis as has been witnessed during the recent COVID-19 outbreak. Traditional methods relying on qPCR or sequencing are difficult to implement in settings with limited resources necessitating the development of accurate alternative testing strategies that perform robustly. Here, we present FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that employs a direct Cas9 based enzymatic readout for detecting nucleotide sequences and identifying nucleobase identity without the requirement of trans-cleavage activity of reporter molecules. We demonstrate that FELUDA is 100% accurate in detecting single nucleotide variants (SNVs) including heterozygous carriers of a mutation and present a simple design strategy in the form of a web-tool, JATAYU, for its implementation. FELUDA is semi quantitative, can be adapted to multiple signal detection platforms and can be quickly designed and deployed for versatile applications such as infectious disease outbreaks like COVID-19. Using a lateral flow readout within 1h, FELUDA shows 100% sensitivity and 97% specificity across all range of viral loads in clinical samples. In combination with RT-RPA and a smartphone application True Outcome Predicted via Strip Evaluation (TOPSE), we present a prototype for FELUDA for CoV-2 detection at home.Single sentence summaryA method to identify nucleotide sequence or nucleobase identity using FnCas9 and its implementation in the rapid and accurate diagnosis of SARS-CoV-2

Detection of pathogenic sequences or variants in DNA and RNA through a point-of-care diagnostic approach is valuable for rapid clinical prognosis. In recent times, CRISPR based detection of nucleic acids has provided an economical and quicker alternative to sequencing-based platforms which are often difficult to implement in the field. Here, we present FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that employs a highly accurate enzymatic readout for detecting nucleotide sequences, identifying nucleobase identity and inferring zygosity with precision. We demonstrate that FELUDA output can be adapted to multiple signal detection platforms and can be quickly designed and deployed for versatile applications including rapid diagnosis during infectious disease outbreaks like COVID-19.

SUMMARYGenome editing using the CRISPR Cas9 system has been used to manipulate eukaryotic DNA and make precise heritable changes. Although the widely usedStreptococcus pyogenesCas9 (SpCas9) and its engineered variants have been efficiently harnessed for numerous gene-editing applications across different platforms, concerns remain, regarding their putative off targeting at multiple loci across the genome. Here we report thatFrancisella novicidaCas9 (FnCas9) shows a very high specificity of binding to its intended targets and negligible binding to off-target loci. The specificity is determined by its minimal binding affinity with DNA when mismatches to the target sgRNA are present in the sgRNA:DNA heteroduplex. FnCas9 produces staggered cleavage, higher HDR rates and very low non-specific genome editing compared to SpCas9. We demonstrate FnCas9 mediated correction of the sickle cell mutation in patient derived iPSCs and propose that it can be used for precise therapeutic genome editing for a wide variety of genetic disorders.SIGNIFICANCE STATEMENTTherapeutic genome editing has been significantly accentuated by the advent of CRISPR based gene correction. However, genomic off-targeting has been a major setback for clinical translation. Although high fidelity versions of Cas9 have been rationally designed, they recognize and bind to off-targets. In this study, we characterize a naturally occurring Cas9 fromFrancisella novicida(FnCas9) that shows negligible binding affinity to off targets differing by one or more mismatches, rendering it highly specific in target recognition and editing. We show that FnCas9 can direct both HDR and NHEJ mediated DNA repair, generates higher rate of HDR and negligible off-target editing. Finally we show its potential in therapeutic genome editing by correcting the sickle cell anemia mutation in patient derived iPSCs.

Somatic variation in DNA can cause cells to deviate from the preordained\ud genomic path in both disease and healthy conditions. Here, using exome\ud sequencing of paired tissue samples, we show that the normal human brain\ud harbors somatic single base variations measuring up to 0.48% of the total\ud variations. Interestingly, about 64% of these somatic variations in the brain are\ud expected to lead to non-synonymous changes, and as much as 87% of these\ud represent G:C>T:A transversion events. Further, the transversion events in the\ud brain were mostly found in the frontal cortex, whereas the corpus callosum from\ud the same individuals harbors the reference genotype. We found a significantly\ud higher amount of 8-OHdG (oxidative stress marker) in the frontal cortex\ud compared to the corpus callosum of the same subjects (pT:A transversions in the cortex. We found significant\ud enrichment for axon guidance and related pathways for genes harbouring\ud somatic variations. This could represent either a directed selection of genetic\ud variations in these pathways or increased susceptibility of some loci towards\ud oxidative stress. This study highlights that oxidative stress possibly influence\ud single nucleotide somatic variations in normal human brain.