Robust Hi-C Maps of Enhancer-Promoter Interactions Reveal the Function of Non-coding Genome in Neural Development and Diseases.

Genome-wide mapping of chromatin interactions at high resolution remains experimentally and computationally challenging. Here we used a low-input "easy Hi-C" protocol to map the 3D genome architecture in human neurogenesis and brain tissues and also demonstrated that a rigorous Hi-C bias-correction pipeline (HiCorr) can significantly improve the sensitivity and robustness of Hi-C loop identification at sub-TAD level, especially the enhancer-promoter (E-P) interactions. We used HiCorr to compare the high-resolution maps of chromatin interactions from 10 tissue or cell types with a focus on neurogenesis and brain tissues. We found that dynamic chromatin loops are better hallmarks for cellular differentiation than compartment switching. HiCorr allowed direct observation of cell-type- and differentiation-specific E-P aggregates spanning large neighborhoods, suggesting a mechanism that stabilizes enhancer contacts during development. Interestingly, we concluded that Hi-C loop outperforms eQTL in explaining neurological GWAS results, revealing a unique value of high-resolution 3D genome maps in elucidating the disease etiology.

Phase-separated DropCRISPRa platform for efficient gene activation in mammalian cells and mice.

Liquid-liquid phase separation (LLPS) plays a critical role in regulating gene transcription via the formation of transcriptional condensates. However, LLPS has not been reported to be engineered as a tool to activate endogenous gene expression in mammalian cells or in vivo. Here, we developed a droplet-forming CRISPR (clustered regularly interspaced short palindromic repeats) gene activation system (DropCRISPRa) to activate transcription with high efficiency via combining the CRISPR-SunTag system with FETIDR-AD fusion proteins, which contain an N-terminal intrinsically disordered region (IDR) of a FET protein (FUS or TAF15) and a transcription activation domain (AD, VP64/P65/VPR). In this system, the FETIDR-AD fusion protein formed phase separation condensates at the target sites, which could recruit endogenous BRD4 and RNA polymerase II with an S2 phosphorylated C-terminal domain (CTD) to enhance transcription elongation. IDR-FUS9Y>S and IDR-FUSG156E, two mutants with deficient and aberrant phase separation respectively, confirmed that appropriate phase separation was required for efficient gene activation. Further, the DropCRISPRa system was compatible with a broad set of CRISPR-associated (Cas) proteins and ADs, including dLbCas12a, dAsCas12a, dSpCas9 and the miniature dUnCas12f1, and VP64, P65 and VPR. Finally, the DropCRISPRa system could activate target genes in mice. Therefore, this study provides a robust tool to activate gene expression for foundational research and potential therapeutics.

Generation of Human Embryonic Stem Cell-Derived Lung Organoids for Modeling Infection and Replication Differences between Human Adenovirus Types 3 and 55 and Evaluating Potential Antiviral Drugs.

Human adenoviruses type 3 (HAdV-3) and type 55 (HAdV-55) are frequently encountered, highly contagious respiratory pathogens with high morbidity rate. In contrast to HAdV-3, one of the most predominant types in children, HAdV-55 is a reemergent pathogen associated with more severe community-acquired pneumonia (CAP) in adults, especially in military camps. However, the infectivity and pathogenicity differences between these viruses remain unknown as in vivo models are not available. Here, we report a novel system utilizing human embryonic stem cells-derived 3-dimensional airway organoids (hAWOs) and alveolar organoids (hALOs) to investigate these two viruses. Firstly, HAdV-55 replicated more robustly than HAdV-3. Secondly, cell tropism analysis in hAWOs and hALOs by immunofluorescence staining revealed that HAdV-55 infected more airway and alveolar stem cells (basal and AT2 cells) than HAdV-3, which may lead to impairment of self-renewal functions post-injury and the loss of cell differentiation in lungs. Additionally, the viral life cycles of HAdV-3 and -55 in organoids were also observed using Transmission Electron Microscopy. This study presents a useful pair of lung organoids for modeling infection and replication differences between respiratory pathogens, illustrating that HAdV-55 has relatively higher replication efficiency and more specific cell tropism in human lung organoids than HAdV-3, which may result in relatively higher pathogenicity and virulence of HAdV-55 in human lungs. The model system is also suitable for evaluating potential antiviral drugs, as demonstrated with cidofovir. IMPORTANCE Human adenovirus (HAdV) infections are a major threat worldwide. HAdV-3 is one of the most predominant respiratory pathogen types found in children. Many clinical studies have reported that HAdV-3 causes less severe disease. In contrast, HAdV-55, a reemergent acute respiratory disease pathogen, is associated with severe community-acquired pneumonia in adults. Currently, no ideal in vivo models are available for studying HAdVs. Therefore, the mechanism of infectivity and pathogenicity differences between human adenoviruses remain unknown. In this study, a useful pair of 3-dimensional (3D) airway organoids (hAWOs) and alveolar organoids (hALOs) were developed to serve as a model. The life cycles of HAdV-3 and HAdV-55 in these human lung organoids were documented for the first time. These 3D organoids harbor different cell types, which are similar to the ones found in humans. This allows for the study of the natural target cells for infection. The finding of differences in replication efficiency and cell tropism between HAdV-55 and -3 may provide insights into the mechanism of clinical pathogenicity differences between these two important HAdV types. Additionally, this study provides a viable and effective in vitro tool for evaluating potential anti-adenoviral treatments.

Short cell-penetration peptide conjugated bioreducible polymer enhances gene editing of CRISPR system.

CRISPR-based gene therapy offers precise targeting and specific editing of disease-related gene sequences, potentially yielding long-lasting treatment effects. However, efficient delivery remains a significant challenge for its widespread application. In this study, we design a novel short peptide-conjugated bioreducible polymer named TSPscp as a safe and effective delivery vector for the CRISPR system. Our results show that TSPscp markedly boosts transcriptional activation and genome editing activities of multiple CRISPR systems as confirmed by decomposition-seq and Deep-seq, which is resulted from its capability in facilitating delivery of plasmid DNA by promoting cellular uptake and lysosomal escape. Additionally, TSPscp further enhances genome editing of CRISPR by delivery of minicircle DNA, a condensed form of regular plasmid DNA. More importantly, TSPscp significantly improves delivery and genome editing of CRISPR system in vivo. In summary, our study highlights TSPscp as a promising delivery tool for CRISPR applications in vivo.

Risk single-nucleotide polymorphism-mediated enhancer-promoter interaction drives keloids through long noncoding RNA down expressed in keloids.

BACKGROUND: Keloids represent one extreme of aberrant dermal wound healing and are characterized by fibroblast hyperproliferation and excessive deposition of extracellular matrix. Genetics is a major factor for predisposition to keloids and genome-wide association study has identified a single-nucleotide polymorphism (SNP) rs873549 at 1q41 as a susceptibility locus. The SNP rs873549, and the SNPs in strong linkage disequilibrium (LD) with rs873549, may be involved in keloid development. However, the functional significance of these SNPs in keloid pathogenesis remains elusive. OBJECTIVES: To investigate the function and mechanism of SNP rs873549 and the SNPs in strong LD with rs873549 in keloids. METHODS: SNPs in strong LD with rs873549 were analysed using Haploview. The expression levels of the genes near the susceptibility locus were analysed using quantitative real-time polymerase chain reaction. The interaction between rs1348270-containing enhancer and the long noncoding RNA down expressed in keloids (DEIK) (formerly RP11-400N13.1) promoter in fibroblasts was investigated using chromosome conformation capture. The enhancer activity of the rs1348270 locus was evaluated using luciferase reporter assay. Knockdown experiments were used to explore the function of DEIK in keloids. RNA-Seq was performed to investigate the mechanism by which DEIK regulates the expression of collagens POSTN and COMP. RESULTS: rs1348270, an enhancer-located SNP in strong LD with rs873549, mediated looping with the promoter of DEIK. The risk variant was associated with decreased enhancer-promoter interaction and DEIK down-expression in keloids. Mechanistically, downregulation of DEIK increased the expression of collagens POSTN and COMP through upregulating BMP2. Furthermore, correlation analysis revealed that DEIK expression was inversely correlated with BMP2, POSTN and COMP expression in both keloid and normal fibroblasts. CONCLUSIONS: Our findings suggest that the risk variant rs1348270 is located in an enhancer and is associated with the downregulation of DEIK in keloids, and that downregulation of DEIK increases the expression of collagens POSTN and COMP through BMP2 in keloid fibroblasts. These findings will help to provide a more thorough understanding of the role played by genetic factors in keloid development and may lead to new strategies for screening and therapy in keloid-susceptible populations.

MiniCAFE, a CRISPR/Cas9-based compact and potent transcriptional activator, elicits gene expression in vivo.

CRISPR-mediated gene activation (CRISPRa) is a promising therapeutic gene editing strategy without inducing DNA double-strand breaks (DSBs). However, in vivo implementation of these CRISPRa systems remains a challenge. Here, we report a compact and robust miniCas9 activator (termed miniCAFE) for in vivo activation of endogenous target genes. The system relies on recruitment of an engineered minimal nuclease-null Cas9 from Campylobacter jejuni and potent transcriptional activators to a target locus by a single guide RNA. It enables robust gene activation in human cells even with a single DNA copy and is able to promote lifespan of Caenorhabditis elegans through activation of longevity-regulating genes. As proof-of-concept, delivered within an all-in-one adeno-associated virus (AAV), miniCAFE can activate Fgf21 expression in the liver and regulate energy metabolism in adult mice. Thus, miniCAFE holds great therapeutic potential against human diseases.

Engineered Cas12a-Plus nuclease enables gene editing with enhanced activity and specificity.

BACKGROUND: The CRISPR-Cas12a (formerly Cpf1) system is a versatile gene-editing tool with properties distinct from the broadly used Cas9 system. Features such as recognition of T-rich protospacer-adjacent motif (PAM) and generation of sticky breaks, as well as amenability for multiplex editing in a single crRNA and lower off-target nuclease activity, broaden the targeting scope of available tools and enable more accurate genome editing. However, the widespread use of the nuclease for gene editing, especially in clinical applications, is hindered by insufficient activity and specificity despite previous efforts to improve the system. Currently reported Cas12a variants achieve high activity with a compromise of specificity. Here, we used structure-guided protein engineering to improve both editing efficiency and targeting accuracy of Acidaminococcus sp. Cas12a (AsCas12a) and Lachnospiraceae bacterium Cas12a (LbCas12a). RESULTS: We created new AsCas12a variant termed "AsCas12a-Plus" with increased activity (1.5~2.0-fold improvement) and specificity (reducing off-targets from 29 to 23 and specificity index increased from 92% to 94% with 33 sgRNAs), and this property was retained in multiplex editing and transcriptional activation. When used to disrupt the oncogenic BRAFV600E mutant, AsCas12a-Plus showed less off-target activity while maintaining comparable editing efficiency and BRAFV600E cancer cell killing. By introducing the corresponding substitutions into LbCas12a, we also generated LbCas12a-Plus (activity improved ~1.1-fold and off-targets decreased from 20 to 12 while specificity index increased from 78% to 89% with 15 sgRNAs), suggesting this strategy may be generally applicable across Cas12a orthologs. We compared Cas12a-Plus, other variants described in this study, and the reported enCas12a-HF, enCas12a, and Cas12a-ultra, and found that Cas12a-Plus outperformed other variants with a good balance for enhanced activity and improved specificity. CONCLUSIONS: Our discoveries provide alternative AsCas12a and LbCas12a variants with high specificity and activity, which expand the gene-editing toolbox and can be more suitable for clinical applications.

Enhancing site-specific DNA integration by a Cas9 nuclease fused with a DNA donor-binding domain.

The CRISPR/Cas system is widely used for genome editing. However, robust and targeted insertion of a DNA segment remains a challenge. Here, we present a fusion nuclease (Cas9-N57) to enhance site-specific DNA integration via a fused DNA binding domain of Sleeping Beauty transposase to tether the DNA segment to the Cas9/sgRNA complex. The insertion was unidirectional and specific, and DNA fragments up to 12 kb in length were successfully integrated. As a test of the system, Cas9-N57 mediated the insertion of a CD19-specific chimeric antigen receptor (CD19-CAR) cassette into the AAVS1 locus in human T cells, and induced intrahepatic cholangiocarcinoma in mice by simultaneously mediating the insertion of oncogenic KrasG12D into the Rosa26 locus and disrupting Trp53 and Pten. Moreover, the nuclease-N57 fusion proteins based on AsCpf1 (AsCas12a) and CjCas9 exhibited similar activity. These findings demonstrate that CRISPR-associated nuclease-N57 protein fusion is a powerful tool for targeted DNA insertion and holds great potential for gene therapy applications.

A Biomimetic Drug Delivery System by Integrating Grapefruit Extracellular Vesicles and Doxorubicin-Loaded Heparin-Based Nanoparticles for Glioma Therapy.

Existing nanoparticle-mediated drug delivery systems for glioma systemic chemotherapy remain a great challenge due to poor delivery efficiency resulting from the blood brain barrier/blood-(brain tumor) barrier (BBB/BBTB) and insufficient tumor penetration. Here, we demonstrate a distinct design by patching doxorubicin-loaded heparin-based nanoparticles (DNs) onto the surface of natural grapefruit extracellular vesicles (EVs), to fabricate biomimetic EV-DNs, achieving efficient drug delivery and thus significantly enhancing antiglioma efficacy. The patching strategy allows the unprecedented 4-fold drug loading capacity compared to traditional encapsulation for EVs. The biomimetic EV-DNs are enabled to bypass BBB/BBTB and penetrate into glioma tissues by receptor-mediated transcytosis and membrane fusion, greatly promoting cellular internalization and antiproliferation ability as well as extending circulation time. We demonstrate that a high-abundance accumulation of EV-DNs can be detected at glioma tissues, enabling the maximal brain tumor uptake of EV-DNs and great antiglioma efficacy in vivo.

Get ready for the CRISPR/Cas system: A beginner's guide to the engineering and design of guide RNAs.

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a state-of-the-art tool for versatile genome editing that has advanced basic research dramatically, with great potential for clinic applications. The system consists of two key molecules: a CRISPR-associated (Cas) effector nuclease and a single guide RNA. The simplicity of the system has enabled the development of a wide spectrum of derivative methods. Almost any laboratory can utilize these methods, although new users may initially be confused when faced with the potentially overwhelming abundance of choices. Cas nucleases and their engineering have been systematically reviewed previously. In the present review, we discuss single guide RNA engineering and design strategies that facilitate more efficient, more specific and safer gene editing.

A Safety Checkpoint to Eliminate Cancer Risk of the Immune Evasive Cells Derived from Human Embryonic Stem Cells.

Human embryonic stem cells (hESCs) hold great promise in the regenerative therapy of many currently untreatable human diseases. One of the key bottlenecks is the immune rejection of hESC-derived allografts by the recipient. To overcome this challenge, we have established new approaches to induce immune protection of hESC-derived allografts through the coexpression of immune suppressive molecules CTLA4-Ig and PD-L1. However, this in turn raises a safety concern of cancer risk because these hESC-derived cells can evade immune surveillance. To address this safety concern, we developed a safety checkpoint so that the immune evasive hESC-derived cells in the graft can be effectively eliminated if any cellular transformation is detected. In this context, we knock-in the suicidal gene herpes simplex virus thymidine kinase (HSVTK) into the constitutive HPRT locus of CP hESCs (knock-in hESCs expressing CTLA4-Ig and PD-L1), denoted CPTK hESCs. Employing humanized mice (Hu-mice) reconstituted with human immune system, we demonstrated that the CPTK hESC-derived cells are protected from immune rejection. In addition, CPTK hESC-derived cells can be efficiently eliminated in vitro and in vivo with FDA approved TK-targeting drug ganciclovir. Therefore, this new safety checkpoint improves the feasibility to use the immune evasive hESC-derived cells for regenerative medicine. Stem Cells 2017;35:1154-1161.

Immunogenicity of induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs), reprogrammed from somatic cells with defined factors, hold great promise for regenerative medicine as the renewable source of autologous cells. Whereas it has been generally assumed that these autologous cells should be immune-tolerated by the recipient from whom the iPSCs are derived, their immunogenicity has not been vigorously examined. We show here that, whereas embryonic stem cells (ESCs) derived from inbred C57BL/6 (B6) mice can efficiently form teratomas in B6 mice without any evident immune rejection, the allogeneic ESCs from 129/SvJ mice fail to form teratomas in B6 mice due to rapid rejection by recipients. B6 mouse embryonic fibroblasts (MEFs) were reprogrammed into iPSCs by either retroviral approach (ViPSCs) or a novel episomal approach (EiPSCs) that causes no genomic integration. In contrast to B6 ESCs, teratomas formed by B6 ViPSCs were mostly immune-rejected by B6 recipients. In addition, the majority of teratomas formed by B6 EiPSCs were immunogenic in B6 mice with T cell infiltration, and apparent tissue damage and regression were observed in a small fraction of teratomas. Global gene expression analysis of teratomas formed by B6 ESCs and EiPSCs revealed a number of genes frequently overexpressed in teratomas derived from EiPSCs, and several such gene products were shown to contribute directly to the immunogenicity of the B6 EiPSC-derived cells in B6 mice. These findings indicate that, in contrast to derivatives of ESCs, abnormal gene expression in some cells differentiated from iPSCs can induce T-cell-dependent immune response in syngeneic recipients. Therefore, the immunogenicity of therapeutically valuable cells derived from patient-specific iPSCs should be evaluated before any clinic application of these autologous cells into the patients.

Dynamic pathological analysis reveals a protective role against skin fibrosis for TREM2-dependent macrophages.

Rationale: Systemic sclerosis (SSc) is a chronic and incurable autoimmune disease with high mortality rates, and skin fibrosis is one of distinguishing hallmarks in the pathogenesis. However, macrophage heterogeneity regulating skin fibrosis remain largely unknown. Methods: We established mouse disease model and performed single-cell RNA-sequencing (scRNA-seq) to resolve the dynamic and heterogenous characteristics of macrophages in skin fibrosis, and the role of TREM2-dependent macrophages in the pathological process was investigated using knockout mice and intraperitoneal transferring TREM2+ macrophages combining with functional assays. Results: We show that TREM2-expressing macrophages (TREM2+ MФs) accumulate in injured skin of mice treated by bleomycin (BLM) and human SSc, and their gene signatures and functional pathways are identified in the course of disease. Genetic ablation of Trem2 in mice globally accelerates and aggravates skin fibrosis, whereas transferring TREM2hi macrophages improves and alleviates skin fibrosis. Amazingly, we found that disease-associated TREM2+ MФs in skin fibrosis exhibit overlapping signatures with fetal skin counterparts in mice and human to maintain skin homeostasis, but each has merits in skin remodeling and development respectively. Conclusion: This study identifies that TREM2 acts as a functional molecule and a major signaling by which macrophage subpopulations play a protective role against fibrosis, and disease-associated TREM2+ MФs in skin fibrosis might undergo a fetal-like reprogramming similar to fetal skin counterparts.

A scalable approach to prevent teratoma formation of human embryonic stem cells.

As the renewable source of all cell types in the body, human embryonic stem cells (hESCs) hold great promise for human cell therapy. However, one major bottleneck that hinders the clinic application of hESCs is that hESCs remaining with their differentiated derivatives pose cancer risk by forming teratomas after transplantation. NANOG is a critical pluripotency factor specifically expressed in hESCs but rarely in their differentiated derivatives. By introducing a hyperactive variant of herpes simplex virus thymidine kinase gene into the 3'-untranslated region of the endogenous NANOG gene of hESCs through homologous recombination, we developed a safe and highly scalable approach to efficiently eliminate the teratoma risk associated with hESCs without apparent negative impact on their differentiated cell types. As thymidine kinase is widely used in human gene therapy trials and is the therapeutic target of U. S. Food and Drug Administration-approved drugs, our strategy could be effectively applied to the clinic development of hESC-based human cell therapy.

Relief of Extracellular Matrix Deposition Repression by Downregulation of IRF1-Mediated TWEAK/Fn14 Signaling in Keloids.

Keloids represent a fibrotic disorder characterized by the excessive deposition of extracellular matrix (ECM). However, the mechanisms through which ECM deposition in keloids is regulated remain elusive. In this study, we found that the expression of both TWEAK and its cognate receptor Fn14 was significantly downregulated in keloids and that TWEAK/Fn14 signaling repressed the expression of ECM-related genes in keloid fibroblasts. The IRF1 gene was essential for this repression, and the TWEAK/Fn14 downstream transcription factor p65 directly bound to the promoter of the IRF1 gene and induced its expression. Furthermore, in patients with keloid, the expression of TWEAK and Fn14 was negatively correlated with that of ECM genes and positively correlated with that of IRF1. These observations indicate that relief of TWEAK/Fn14/IRF1-mediated ECM deposition repression contributes to keloid pathogenesis, and the identified mechanism and related molecules provide potential targets for keloid treatment in the future.

SLC35E1 promotes keratinocyte proliferation in psoriasis by regulating zinc homeostasis.

Keratinocyte hyperproliferation is a key pathogenic factor in psoriasis. However, the mechanisms that regulate keratinocyte hyperproliferation in this condition remain unclear. Here, we found that SLC35E1 was highly expressed in keratinocytes of patients with psoriasis and that Slc35e1-/- mice displayed a less severe imiquimod (IMQ)-induced psoriasis-like phenotype than their wild-type siblings. In addition, SLC35E1 deficiency inhibited keratinocyte proliferation in both mice and cultured cells. On a molecular level, SLC35E1 was found to regulate zinc ion concentrations and subcellular localization, while zinc ion chelation reversed the IMQ-induced psoriatic phenotype in Slc35e1-/- mice. Meanwhile, epidermal zinc ion levels were decreased in patients with psoriasis and zinc ion supplementation alleviated the psoriatic phenotype in an IMQ-induced mouse model of psoriasis. Our results indicated that SLC35E1 can promote keratinocyte proliferation by regulating zinc ion homeostasis and zinc ion supplementation has potential as a therapy for psoriasis.

Engineered circular guide RNAs boost CRISPR/Cas12a- and CRISPR/Cas13d-based DNA and RNA editing.

BACKGROUND: The CRISPR/Cas12a and CRISPR/Cas13d systems are widely used for fundamental research and hold great potential for future clinical applications. However, the short half-life of guide RNAs (gRNAs), particularly free gRNAs without Cas nuclease binding, limits their editing efficiency and durability. RESULTS: Here, we engineer circular free gRNAs (cgRNAs) to increase their stability, and thus availability for Cas12a and Cas13d processing and loading, to boost editing. cgRNAs increases the efficiency of Cas12a-based transcription activators and genomic DNA cleavage by approximately 2.1- to 40.2-fold for single gene editing and 1.7- to 2.1-fold for multiplexed gene editing than their linear counterparts, without compromising specificity, across multiple sites and cell lines. Similarly, the RNA interference efficiency of Cas13d is increased by around 1.8-fold. In in vivo mouse liver, cgRNAs are more potent in activating gene expression and cleaving genomic DNA. CONCLUSIONS: CgRNAs enable more efficient programmable DNA and RNA editing for Cas12a and Cas13d with broad applicability for fundamental research and gene therapy.

Generation of human embryonic stem cell-derived lung organoids.

This protocol describes how to generate lung organoids from human embryonic stem cells. Lung organoids form by self-assembly in Matrigel and contain lung epithelial cell types. The protocol presented in this study is simple and only uses 6 cytokines or small molecules. This protocol provides a promising tool to study human lung development, drug screening, regeneration, and disease modeling in vitro. For complete details on the use and execution of this protocol, please refer to Chen et al. (2018).

Live-cell imaging of genomic loci with Cas9 variants.

BACKGROUND: Endonuclease-deactivated clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (dCas9) has been repurposed for live-cell imaging of genomic loci. Engineered or evolved dCas9 variants have been developed to increase the applicability of the CRISPR/dCas9 system. However, there have been no systematic comparisons of these dCas9 variants in terms of their performance in the visualization of genomic loci. MAIN METHODS AND MAJOR RESULTS: Here we demonstrate that dSpCas9 and its variants deSpCas9(1.1), dSpCas9-HF1, devoCas9, and dxCas9(3.7) can be used for CRISPR-based live-cell genomic imaging. dSpCas9 had the greatest utility, with a high labeling efficiency of repetitive sequences-including those with a low number of repeats-and good compatibility with target RNA sequences at the MUC4 locus that varied in length from 13 to 23 nucleotides. We combined CRISPR-Tag with the dSpCas9 imaging system to observe the dynamics of the Tet promoter and found that its movement was restricted when it was active. CONCLUSIONS AND IMPLICATIONS: These novel Cas9 variants provide a new set of tools for investigating the spatiotemporal regulation of gene expression through live imaging of genomic sites.