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CRISPR-Cas9 has emerged as a major genome manipulation tool. As Cas9 can cause off-target effects, several methods for controlling the expression of CRISPR systems were developed. Recent studies have shown that CRISPR activity could be controlled by sensing expression levels of endogenous transcripts. This is particularly interesting, as endogenous RNAs could harbor important information about the cell type, disease state, and environmental challenges cells are facing. Single-guide RNA (sgRNA) engineering played a major role in the development of RNA-responsive CRISPR systems. Following further optimizations, RNA-responsive sgRNAs could enable the development of novel therapeutic and research applications. This review introduces engineering strategies that could be employed to modify Streptococcus pyogenes sgRNAs with a focus on recent advances made toward the development of RNA-responsive sgRNAs. Future directions and potential applications of these technologies are also discussed.

Contains fulltext : 286832.pdf (Publisher’s version ) (Open Access) BACKGROUND: Direct evaluation of vascular inflammation in patients with COVID-19 would facilitate more efficient trials of new treatments and identify patients at risk of long-term complications who might respond to treatment. We aimed to develop a novel artificial intelligence (AI)-assisted image analysis platform that quantifies cytokine-driven vascular inflammation from routine CT angiograms, and sought to validate its prognostic value in COVID-19. METHODS: For this prospective outcomes validation study, we developed a radiotranscriptomic platform that uses RNA sequencing data from human internal mammary artery biopsies to develop novel radiomic signatures of vascular inflammation from CT angiography images. We then used this platform to train a radiotranscriptomic signature (C19-RS), derived from the perivascular space around the aorta and the internal mammary artery, to best describe cytokine-driven vascular inflammation. The prognostic value of C19-RS was validated externally in 435 patients (331 from study arm 3 and 104 from study arm 4) admitted to hospital with or without COVID-19, undergoing clinically indicated pulmonary CT angiography, in three UK National Health Service (NHS) trusts (Oxford, Leicester, and Bath). We evaluated the diagnostic and prognostic value of C19-RS for death in hospital due to COVID-19, did sensitivity analyses based on dexamethasone treatment, and investigated the correlation of C19-RS with systemic transcriptomic changes. FINDINGS: Patients with COVID-19 had higher C19-RS than those without (adjusted odds ratio [OR] 2·97 [95% CI 1·43-6·27], p=0·0038), and those infected with the B.1.1.7 (alpha) SARS-CoV-2 variant had higher C19-RS values than those infected with the wild-type SARS-CoV-2 variant (adjusted OR 1·89 [95% CI 1·17-3·20] per SD, p=0·012). C19-RS had prognostic value for in-hospital mortality in COVID-19 in two testing cohorts (high [≥6·99] vs low [

Olfactory ensheathing cells (OECs) are neural crest-derived glia that ensheath bundles of olfactory axons from their peripheral origins in the olfactory epithelium to their central targets in the olfactory bulb. We took an unbiased laser microdissection and differential RNA-seq approach, validated by in situ hybridization, to identify candidate molecular mechanisms underlying mouse OEC development and differences with the neural crest-derived Schwann cells developing on other peripheral nerves. We identified 25 novel markers for developing OECs in the olfactory mucosa and/or the olfactory nerve layer surrounding the olfactory bulb, of which 15 were OEC-specific (that is, not expressed by Schwann cells). One pan-OEC-specific gene, Ptprz1, encodes a receptor-like tyrosine phosphatase that blocks oligodendrocyte differentiation. Mutant analysis suggests Ptprz1 may also act as a brake on OEC differentiation, and that its loss disrupts olfactory axon targeting. Overall, our results provide new insights into OEC development and the diversification of neural crest-derived glia.

Immunotherapies have recently gained traction as highly effective therapies in a subset of late-stage cancers. Unfortunately, only a minority of patients experience the remarkable benefits of immunotherapies, whilst others fail to respond or even come to harm through immune related adverse events. For immunotherapies within the PD-1/PD-L1 inhibitor class, patient stratification is currently performed using tumor (tissue-based) PD-L1 expression. However, PD-L1 is an accurate predictor of response in only ∼30% of cases. There is pressing need for more accurate biomarkers for immunotherapy response prediction.We sought to identify peripheral blood biomarkers, predictive of response to immunotherapies against lung cancer, based on whole blood microRNA profiling. Using three well characterized cohorts consisting of a total of 334 stage IV NSCLC patients, we have defined a 5 microRNA risk score (miRisk) that is predictive of overall survival following immunotherapy in training and independent validation (HR 2.40, 95% CI 1.37-4.19; P < 0.01) cohorts. We have traced the signature to a myeloid origin and performed miRNA target prediction to make a direct mechanistic link to the PD-L1 signalling pathway and PD-L1 itself. The miRisk score offers a potential blood-based companion diagnostic for immunotherapy that outperforms tissue-based PD-L1 staining.

In vertebrates, neural crest cells (NCCs) are a multipotent embryonic population generating both neural/neuronal and mesenchymal derivatives, and thus the neural crest (NC) is often referred to as the fourth germ layer. NC development is a dynamic process, where NCCs possess substantial plasticity in transcriptional and epigenomic profiles. Recent technical advances in single-cell and low-input sequencing have empowered fine-resolution characterisation of NC development. In this review, we summarise the latest models underlying NC-plasticity acquirement and cell-fate restriction, outline the connections between NC plasticity and NC-derived cancer and envision the new opportunities in studying NC plasticity and its link to cancer.

14 pages, 8 figures, supplementary information https://doi.org/10.1038/s41588-022-01089-w.-- Data availability: Raw and aligned sequencing data are available at https://danio-code.zfin.org/dataExport/. The raw sequencing data produced for this study are available on the European Bioinformatics Institute (EBI) European Nucleotide Archive (ENA) under study numbers PRJNA824720, PRJNA821001, PRJNA821088, PRJNA821148 and PRJNA821034. Annotation tracks are available at http://genome.ucsc.edu/cgi-bin/hgTracks?db=danRer10&hubUrl=https://danio-code.zfin.org/trackhub/DANIO-CODE.hub.txt (danRer10) and http://genome.ucsc.edu/cgi-bin/hgTracks?db=danRer11&hubUrl=https://danio-code.zfin.org/trackhub/DANIO-CODE.hub.txt (danRer11). Code availability: The processing pipelines for the individual assays are available at https://gitlab.com/danio-code. The code used for the analysis is available at https://github.com/DANIO-CODE/DANIO-CODE_Data_analysis (https://doi.org/10.5281/zenodo.6424702). The script to generate the TrackHub is available at https://gitlab.com/danio-code/TrackHub, Zebrafish, a popular organism for studying embryonic development and for modeling human diseases, has so far lacked a systematic functional annotation program akin to those in other animal models. To address this, we formed the international DANIO-CODE consortium and created a central repository to store and process zebrafish developmental functional genomic data. Our data coordination center (https://danio-code.zfin.org) combines a total of 1,802 sets of unpublished and re-analyzed published genomic data, which we used to improve existing annotations and show its utility in experimental design. We identified over 140,000 cis-regulatory elements throughout development, including classes with distinct features dependent on their activity in time and space. We delineated the distinct distance topology and chromatin features between regulatory elements active during zygotic genome activation and those active during organogenesis. Finally, we matched regulatory elements and epigenomic landscapes between zebrafish and mouse and predicted functional relationships between them beyond sequence similarity, thus extending the utility of zebrafish developmental genomics to mammals, We thank our main funders, the Horizon 2020 MSCA-ITN project ZENCODE-ITN by the European Commission to F.M., B.L., C.O.D., J.M.V. and B.P. (GA no: 643062), BBSRC support (DanioPeaks, P61715) to B.L., F.M. and F.C.W., Wellcome Trust (Joint-Investigator award 106955/Z/15/Z) to F.M. and B.L. and AQUA-FAANG (Horizon 2020, GA 817923) to B.L., D.B. and F.M. and BBSRC (BB/R015457/1) to F.vE and Key Special Project for Introduced Talents Team to Z.C. (GML2019ZD0401) and PrecisionTox project by the European Commission (GA no: 965406), With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)

Zebrafish, a popular model for embryonic development and for modelling human diseases, has so far lacked a systematic functional annotation programme akin to those in other animal models. To address this, we formed the international DANIO-CODE consortium and created the first central repository to store and process zebrafish developmental functional genomic data. Our Data Coordination Center (https://danio-code.zfin.org) combines a total of 1,802 sets of unpublished and reanalysed published genomics data, which we used to improve existing annotations and show its utility in experimental design. We identified over 140,000 cis-regulatory elements in development, including novel classes with distinct features dependent on their activity in time and space. We delineated the distinction between regulatory elements active during zygotic genome activation and those active during organogenesis, identifying new aspects of how they relate to each other. Finally, we matched regulatory elements and epigenomic landscapes between zebrafish and mouse and predict functional relationships between them beyond sequence similarity, extending the utility of zebrafish developmental genomics to mammals.

The epiblast of vertebrate embryos is comprised of neural and non-neural ectoderm, with the border territory at their intersection harbouring neural crest and cranial placode progenitors. Here we profile avian epiblast cells as a function of time using single-cell RNA-seq to define transcriptional changes in the emerging ‘neural plate border’. The results reveal gradual establishment of heterogeneous neural plate border signatures, including novel genes that we validate by fluorescent in situ hybridisation. Developmental trajectory analysis shows that segregation of neural plate border lineages only commences at early neurulation, rather than at gastrulation as previously predicted. We find that cells expressing the prospective neural crest marker Pax7 contribute to multiple lineages, and a subset of premigratory neural crest cells shares a transcriptional signature with their border precursors. Together, our results suggest that cells at the neural plate border remain heterogeneous until early neurulation, at which time progenitors become progressively allocated toward defined lineages.

The neural crest (NC) is an embryonic cell population that contributes to key vertebrate-specific features including the craniofacial skeleton and peripheral nervous system. Here we examine the transcriptional and epigenomic profiles of NC cells in the sea lamprey, in order to gain insight into the ancestral state of the NC gene regulatory network (GRN). Transcriptome analyses identify clusters of co-regulated genes during NC specification and migration that show high conservation across vertebrates but also identify transcription factors (TFs) and cell-adhesion molecules not previously implicated in NC migration. ATAC-seq analysis uncovers an ensemble of cis-regulatory elements, including enhancers of Tfap2B, SoxE1 and Hox-α2 validated in the embryo. Cross-species deployment of lamprey elements identifies the deep conservation of lamprey SoxE1 enhancer activity, mediating homologous expression in jawed vertebrates. Our data provide insight into the core GRN elements conserved to the base of the vertebrates and expose others that are unique to lampreys., An understanding of the ancestral state of the neural crest (NC) gene regulatory network (GRN) gives insight into vertebrate evolution. Here, the authors use transcriptomic and chromatin accessibility analyses of the lamprey NC, as well as cross-species enhancer assays, to identify GRN elements conserved throughout vertebrates.

SummaryUnlike the adult mammalian heart, which has limited regenerative capacity, the zebrafish heart can fully regenerate following injury. Reactivation of cardiac developmental programmes is considered key to successfully regenerating the heart, yet the regulatory elements underlying the response triggered upon injury and during development remain elusive. Organ-wide activation of the epicardium is essential for zebrafish heart regeneration and is considered a potential regenerative source to target in the mammalian heart. Here we compared the transcriptome and epigenome of the developing and regenerating zebrafish epicardium by integrating gene expression profiles with open chromatin ATAC-seq data. By generating gene regulatory networks associated with epicardial development and regeneration, we inferred genetic programmes driving each of these processes, which were largely distinct. We identified wt1a, wt1b, and the AP-1 subunits junbb, fosab and fosb as central regulators of the developing network, whereas hif1ab, zbtb7a, tbx2b and nrf1 featured as putative central regulators of the regenerating epicardial network. By interrogating developmental gene regulatory networks that drive cell-specific transcriptional heterogeneity, we tested novel subpopulation-related epicardial enhancers in vivo. Taken together, our work revealed striking differences between the regulatory blueprint deployed during epicardial development and regeneration. These findings challenge the dogma that heart regeneration is essentially a reactivation of developmental programmes, and provide important insights into epicardial regulation that can assist in developing therapeutic approaches to enable tissue regeneration in the adult mammalian heart.

Treatment of severe COVID-19 is currently limited by clinical heterogeneity and incomplete description of specific immune biomarkers. We present here a comprehensive multi-omic blood atlas for patients with varying COVID-19 severity in an integrated comparison with influenza and sepsis patients versus healthy volunteers. We identify immune signatures and correlates of host response. Hallmarks of disease severity involved cells, their inflammatory mediators and networks, including progenitor cells and specific myeloid and lymphocyte subsets, features of the immune repertoire, acute phase response, metabolism and coagulation. Persisting immune activation involving AP-1/p38MAPK was a specific feature of COVID-19. The plasma proteome enabled sub-phenotyping into patient clusters, predictive of severity and outcome. Systems-based integrative analyses including tensor and matrix decomposition of all modalities revealed feature groupings linked with severity and specificity compared to influenza and sepsis. Our approach and blood atlas will support future drug development, clinical trial design and personalized medicine approaches for COVID-19., Graphical Abstract, A multi-omic analysis of patient blood samples reveals both similarities and specific features of COVID-19 when compared with samples obtained from sepsis or influenza patients which could yield better targetted therapies for severe COVID-19.

Similar to tumor-initiating cells (TICs), minimal residual disease (MRD) is capable of reinitiating tumors and causing recurrence. However, the molecular characteristics of solid tumor MRD cells and drivers of their survival have remained elusive. Here we performed dense multiregion transcriptomics analysis of paired biopsies from 17 ovarian cancer patients before and after chemotherapy. We reveal that while MRD cells share important molecular signatures with TICs, they are also characterized by an adipocyte-like gene expression signature and a portion of them had undergone epithelial-mesenchymal transition (EMT). In a cell culture MRD model, MRD-mimic cells showed the same phenotype and were dependent on fatty acid oxidation (FAO) for survival and resistance to cytotoxic agents. These findings identify EMT and FAO as attractive targets to eradicate MRD in ovarian cancer and make a compelling case for the further testing of FAO inhibitors in treating MRD.

Summary Here, we describe a highly efficient, medium-throughput strategy for cloning and in vivo screening of putative enhancers using the chick embryo. By incorporating 48 unique nanotags for use in NanoString nCounter® across three different fluorescent reporters and developing a rapid and efficient digestion/ligation type IIs restriction enzyme-based cloning protocol, we develop a multiplexed approach for rapidly identifying enhancer activity. For complete details on the use and execution of this protocol, please see Williams et al. (2019)., Graphical abstract, Highlights • Highly efficient, medium-throughput protocol for cloning putative enhancers • Multiplexed, rapid screening strategy for detecting in vivo enhancer activity • Reproducible, non-mosaic validation of spatial and temporal enhancer activity in vivo • Functional conservation allows cross-species enhancer screening, Here, we describe a highly efficient, medium-throughput strategy for cloning and in vivo screening of putative enhancers using the chick embryo. By incorporating 48 unique nanotags for use in NanoString nCounter® across three different fluorescent reporters and developing a rapid and efficient digestion/ligation type IIs restriction enzyme-based cloning protocol, we develop a multiplexed approach for rapidly identifying enhancer activity.

Neural crest ontogeny plays a prominent role in craniofacial development. In this Perspective article, we discuss recent advances to the understanding of mechanisms underlying the cranial neural crest gene regulatory network (cNC-GRN) stemming fromomics-based studies. We briefly summarize how parallel considerations of transcriptome, interactome, and epigenome data significantly elaborated the roles of key players derived from pre-omicsera studies. Furthermore, the growing cohort of cNC multiomics data revealed contribution of the non-coding genomic landscape. As technological improvements are constantly being developed, we reflect on key questions we are poised to address by taking advantage of the unique perspective a multiomics approach has to offer.

Somites arising from paraxial mesoderm are a hallmark of the segmented vertebrate body plan. They form sequentially during axis extension and generate musculoskeletal cell lineages. How paraxial mesoderm becomes regionalised along the axis and how this correlates with dynamic changes of chromatin accessibility and the transcriptome remains unknown. Here, we report a spatiotemporal series of ATAC-seq and RNA-seq along the chick embryonic axis. Footprint analysis shows differential coverage of binding sites for several key transcription factors, including CDX2, LEF1 and members of HOX clusters. Associating accessible chromatin with nearby expressed genes identifies cis-regulatory elements (CRE) for TCF15 and MEOX1. We determine their spatiotemporal activity and evolutionary conservation in Xenopus and human. Epigenome silencing of endogenous CREs disrupts TCF15 and MEOX1 gene expression and recapitulates phenotypic abnormalities of anterior–posterior axis extension. Our integrated approach allows dissection of paraxial mesoderm regulatory circuits in vivo and has implications for investigating gene regulatory networks., How paraxial mesoderm formation and differentiation is regulated is unclear. Here, the authors identify accessible chromatin and gene expression signatures that define different stages of paraxial mesoderm development in the chick and identify CREs important for vertebrate anterior–posterior axis formation.

Neural crest (NC) is a vertebrate-specific population of multipotent embryonic cells predisposed to diverse derivatives along the anteroposterior (A-P) axis. Only cranial NC progenitors give rise to ectomesenchymal cell types, whereas trunk NC is biased for neuronal cell fates. By integrating multimodal single-cell analysis, we provide evidence for divergent embryonic origins of cranial vs. trunk NC that explain this dichotomy. We show that the NC regulator foxd3 is heterogeneously expressed across the A-P axis and identify its specific cranial and trunk autoregulatory enhancers. Whereas cranial-specific enhancer is active in thebona fideNC, the trunk foxd3 autoregulatory element surprisingly marked bipotent tailbud neuromesodermal progenitors (NMps). We integrated NMp single cell epigemomics and trasncriptomics data and for the first time reconstructed anamniote NMp gene regulatory network. Moreover, using pseudotime and developmental trajectory analyses of NMps and NC during normal development and infoxd3mutants, we demonstrate an active role for foxd3 in balancing non-cranial NC and NMp fates during early embryonic development. Strikingly, we show that a portion of posterior NC in the developing zebrafish embryo is derived from the pro-neural NMps. This suggests a common embryonic origin of trunk NC and NM progenitors that is distinct from cranial NC anlage, and elucidates pro-neural bias of trunk NC.

Neural crest (NC) is a vertebrate-specific population of multipotent embryonic cells predisposed to diverse derivatives along the anteroposterior (A-P) axis. Only cranial NC progenitors give rise to ectomesenchymal cell types, whereas trunk NC is biased for neuronal cell fates. By integrating multimodal single-cell analysis, we provide evidence for divergent embryonic origins of cranial vs. trunk NC that explain this dichotomy. We show that the NC regulator neural crest, neuromesodermal progenitors, gene regulatory network, axial progenitors, embryo patterning is heterogeneously expressed across the A-P axis and identify its specific cranial and trunk autoregulatory enhancers. Whereas cranial-specific enhancer is active in the bona fide NC, the trunk neural crest, neuromesodermal progenitors, gene regulatory network, axial progenitors, embryo patterning autoregulatory element surprisingly marked bipotent tailbud neuromesodermal progenitors (NMps). We integrated NMp single cell epigemomics and trasncriptomics data and for the first time reconstructed anamniote NMp gene regulatory network. Moreover, using pseudotime and developmental trajectory analyses of NMps and NC during normal development and inneural crest, neuromesodermal progenitors, gene regulatory network, axial progenitors, embryo patterning mutants, we demonstrate an active role for neural crest, neuromesodermal progenitors, gene regulatory network, axial progenitors, embryo patterning in balancing non-cranial NC and NMp fates during early embryonic development. Strikingly, we show that a portion of posterior NC in the developing zebrafish embryo is derived from the pro-neural NMps. This suggests a common embryonic origin of trunk NC and NM progenitors that is distinct from cranial NC anlage, and elucidates pro-neural bias of trunk NC.

Summary Non-coding mutations at the far end of a large gene desert surrounding the SOX9 gene result in a human craniofacial disorder called Pierre Robin sequence (PRS). Leveraging a human stem cell differentiation model, we identify two clusters of enhancers within the PRS-associated region that regulate SOX9 expression during a restricted window of facial progenitor development at distances up to 1.45 Mb. Enhancers within the 1.45 Mb cluster exhibit highly synergistic activity that is dependent on the Coordinator motif. Using mouse models, we demonstrate that PRS phenotypic specificity arises from the convergence of two mechanisms: confinement of Sox9 dosage perturbation to developing facial structures through context-specific enhancer activity and heightened sensitivity of the lower jaw to Sox9 expression reduction. Overall, we characterize the longest-range human enhancers involved in congenital malformations, directly demonstrate that PRS is an enhanceropathy, and illustrate how small changes in gene expression can lead to morphological variation., Graphical Abstract, Highlights • Extreme long-range enhancer clusters overlap PRS patient mutations at the SOX9 locus • PRS enhancers drive stage-specific SOX9 expression in the cranial neural crest • Mandible development has heightened sensitivity to perturbation of SOX9 gene dosage • Deletion of mouse EC1.45 leads to quantitative changes in mandible morphology, Non-coding mutations over a megabase from SOX9 cause the craniofacial disorder Pierre Robin sequence (PRS). Long et al. leverage a human neural crest model to demonstrate that PRS is caused by loss of extreme long-range enhancers active during a restricted developmental window and explore mechanisms underlying the specificity of disease manifestations.

Summary Chromatin immunoprecipitation with sequencing (ChIP-seq) has been instrumental in understanding transcription factor (TF) binding during gene regulation. ChIP-seq requires specific antibodies against desired TFs, which are not available for numerous species. Here, we describe a tissue-specific biotin ChIP-seq protocol for zebrafish and chicken embryos which utilizes AVI tagging of TFs, permitting their biotinylation by a co-expressed nuclear biotin ligase. Subsequently, biotinylated factors can be precipitated with streptavidin beads, enabling the user to construct TF genome-wide binding landscapes like conventional ChIP-seq methods. For complete details on the use and execution of this protocol, please see Lukoseviciute et al. (2018) and Ling and Sauka-Spengler (2019)., Graphical Abstract, Highlights • Tissue-specific in vivo ChIP for biotinylated DNA-binding proteins of interest • Protocol generates genome-wide binding maps in chicken or zebrafish • ChIP-seq can be performed without antibody usage and cell sorting • Protocol requires a relatively low number of cells as input (100,000–150,000), Chromatin immunoprecipitation with sequencing (ChIP-seq) has been instrumental in understanding transcription factor (TF) binding during gene regulation. ChIP-seq requires specific antibodies against desired TFs, which are not available for numerous species. Here, we describe a tissue-specific biotin ChIP-seq protocol for zebrafish and chicken embryos which utilizes AVI tagging of TFs, permitting their biotinylation by a co-expressed nuclear biotin ligase. Subsequently, biotinylated factors can be precipitated with streptavidin beads, enabling the user to construct TF genome-wide binding landscapes like conventional ChIP-seq methods.

The enteric nervous system (ENS) predominantly originates from vagal neural crest (VNC) cells that emerge from the caudal hindbrain, invade the foregut and populate the gastrointestinal tract. However, the gene regulatory network (GRN) orchestrating the early specification of VNC remains unknown. Using an EdnrB enhancer, we generated a comprehensive temporal map of the chromatin and transcriptional landscape of VNC in the avian model, revealing three VNC cell clusters (neural, neurogenic and mesenchymal), each predetermined epigenetically prior to neural tube delamination. We identify and functionally validate regulatory cores (Sox10/Tfap2B/SoxB/Hbox) mediating each programme and elucidate their combinatorial activities with other spatiotemporally specific transcription factors (bHLH/NR). Our global deconstruction of the VNC-GRN in vivo sheds light on critical early regulatory mechanisms that may influence the divergent neural phenotypes in enteric neuropathies.

Bulk whole genome sequencing (WGS) enables the analysis of tumor evolution but, because of depth limitations, can only identify old mutational events. The discovery of current mutational processes for predicting the tumor's evolutionary trajectory requires dense sequencing of individual clones or single cells. Such studies, however, are inherently problematic because of the discovery of excessive false positive mutations when sequencing picogram quantities of DNA. Data pooling to increase the confidence in the discovered mutations, moves the discovery back in the past to a common ancestor. Here we report a robust whole genome sequencing and analysis pipeline (DigiPico/MutLX) that virtually eliminates all false positive results while retaining an excellent proportion of true positives. Using our method, we identified, for the first time, a hyper-mutation (kataegis) event in a group of ∼30 cancer cells from a recurrent ovarian carcinoma. This was unidentifiable from the bulk WGS data. Overall, we propose DigiPico/MutLX method as a powerful framework for the identification of clone-specific variants at an unprecedented accuracy.