High-throughput Functional Genomics Research Articles

A high-throughput functional genomics workflow based on CRISPR/Cas9-mediated targeted mutagenesis in zebrafish.

The zebrafish is a popular model organism for studying development and disease, and genetically modified zebrafish provide an essential tool for functional genomic studies. Numerous publications have demonstrated the efficacy of gene targeting in zebrafish using CRISPR/Cas9, and they have included descriptions of a variety of tools and methods for guide RNA synthesis and mutant identification. However, most of the published techniques are not readily scalable to increase throughput. We recently described a CRISPR/Cas9-based high-throughput mutagenesis and phenotyping pipeline in zebrafish. Here, we present a complete workflow for this pipeline, including target selection; cloning-free single-guide RNA (sgRNA) synthesis; microinjection; validation of the target-specific activity of the sgRNAs; founder screening to identify germline-transmitting mutations by fluorescence PCR; determination of the exact lesion by Sanger or next-generation sequencing (including software for analysis); and genotyping in the F1 or subsequent generations. Using these methods, sgRNAs can be evaluated in 3 d, zebrafish germline-transmitting mutations can be identified within 3 months and stable lines can be established within 6 months. Realistically, two researchers can target tens to hundreds of genes per year using this protocol.

FlyRNAi.org-the database of the Drosophila RNAi screening center and transgenic RNAi project: 2017 update.

The FlyRNAi database of the Drosophila RNAi Screening Center (DRSC) and Transgenic RNAi Project (TRiP) at Harvard Medical School and associated DRSC/TRiP Functional Genomics Resources website (http://fgr.hms.harvard.edu) serve as a reagent production tracking system, screen data repository, and portal to the community. Through this portal, we make available protocols, online tools, and other resources useful to researchers at all stages of high-throughput functional genomics screening, from assay design and reagent identification to data analysis and interpretation. In this update, we describe recent changes and additions to our website, database and suite of online tools. Recent changes reflect a shift in our focus from a single technology (RNAi) and model species (Drosophila) to the application of additional technologies (e.g. CRISPR) and support of integrated, cross-species approaches to uncovering gene function using functional genomics and other approaches.

Next-Generation High-Throughput Functional Annotation of Microbial Genomes.

ABSTRACTHost infection by microbial pathogens cues global changes in microbial and host cell biology that facilitate microbial replication and disease. The complete maps of thousands of bacterial and viral genomes have recently been defined; however, the rate at which physiological or biochemical functions have been assigned to genes has greatly lagged. The National Institute of Allergy and Infectious Diseases (NIAID) addressed this gap by creating functional genomics centers dedicated to developing high-throughput approaches to assign gene function. These centers require broad-based and collaborative research programs to generate and integrate diverse data to achieve a comprehensive understanding of microbial pathogenesis. High-throughput functional genomics can lead to new therapeutics and better understanding of the next generation of emerging pathogens by rapidly defining new general mechanisms by which organisms cause disease and replicate in host tissues and by facilitating the rate at which functional data reach the scientific community.

Tissue-specific Proteogenomic Analysis of Plutella xylostella Larval Midgut Using a Multialgorithm Pipeline

The diamondback moth, Plutella xylostella (L.), is the major cosmopolitan pest of brassica and other cruciferous crops. Its larval midgut is a dynamic tissue that interfaces with a wide variety of toxicological and physiological processes. The draft sequence of the P. xylostella genome was recently released, but its annotation remains challenging because of the low sequence coverage of this branch of life and the poor description of exon/intron splicing rules for these insects. Peptide sequencing by computational assignment of tandem mass spectra to genome sequence information provides an experimental independent approach for confirming or refuting protein predictions, a concept that has been termed proteogenomics. In this study, we carried out an in-depth proteogenomic analysis to complement genome annotation of P. xylostella larval midgut based on shotgun HPLC-ESI-MS/MS data by means of a multialgorithm pipeline. A total of 876,341 tandem mass spectra were searched against the predicted P. xylostella protein sequences and a whole-genome six-frame translation database. Based on a data set comprising 2694 novel genome search specific peptides, we discovered 439 novel protein-coding genes and corrected 128 existing gene models. To get the most accurate data to seed further insect genome annotation, more than half of the novel protein-coding genes, i.e. 235 over 439, were further validated after RT-PCR amplification and sequencing of the corresponding transcripts. Furthermore, we validated 53 novel alternative splicings. Finally, a total of 6764 proteins were identified, resulting in one of the most comprehensive proteogenomic study of a nonmodel animal. As the first tissue-specific proteogenomics analysis of P. xylostella, this study provides the fundamental basis for high-throughput proteomics and functional genomics approaches aimed at deciphering the molecular mechanisms of resistance and controlling this pest.

Foxtail Mosaic Virus-Induced Gene Silencing in Monocot Plants.

Virus-induced gene silencing (VIGS) is a powerful technique to study gene function in plants. However, very few VIGS vectors are available for monocot plants. Here we report that Foxtail mosaic virus (FoMV) can be engineered as an effective VIGS system to induce efficient silencing of endogenous genes in monocot plants including barley (Hordeum vulgare L.), wheat (Triticum aestivum) and foxtail millet (Setaria italica). This is evidenced by FoMV-based silencing of phytoene desaturase (PDS) and magnesium chelatase in barley, of PDS and Cloroplastos alterados1 in foxtail millet and wheat, and of an additional gene IspH in foxtail millet. Silencing of these genes resulted in photobleached or chlorosis phenotypes in barley, wheat, and foxtail millet. Furthermore, our FoMV-based gene silencing is the first VIGS system reported for foxtail millet, an important C4 model plant. It may provide an efficient toolbox for high-throughput functional genomics in economically important monocot crops.

Secrets of Endometrial Receptivity: Some Are Hidden in Uterine Secretome.

Endometrium, the innermost mucosal layer of the uterus, serves as a lodge for the embryo in eutherian mammals. The endometrium is constituted of various cell types, and each cell type executes specific functions to facilitate embryo implantation and development. It is well established that the endometrium, despite being non-permissive to the embryo for the major period of a menstrual cycle, is irreplaceable in the scheme of events essential for procreation. However, the embryo, before initiating physical contact with the endometrium, encounters the uterine cavity that remains bathed in uterine fluid. Uterine fluid is an admixture of endometrial secretions, plasma transudates, and oviductal fluid. Uterine fluid components are believed to play important roles in immunosuppression and embryo development during peri-implantation period. Uterine fluid is also involved in defense against pathogens, sperm migration, and lubrication of endometrium. The advent of high-throughput functional genomics tools has created enormous opportunities to investigate the uterine fluid for its protein repertoire and modulation during the receptive phase of an endometrial cycle in animals and humans. Towards this, few investigations have been conducted in recent years. The data obtained using non-targetted functional genomics approaches need to be assimilated with the existing information on specific components of uterine fluid. This review compiles existing information on the composition of uterine fluid and its significance in endometrial functions and dysfunctions. Collectively, investigations based on targetted and non-targetted approaches have revealed the presence of several cytokines, growth factors, ions, carbohydrates, and steroids, in human uterine fluid. Detailed investigations of human uterine fluid, especially directed towards the elucidation of functional relevance of different proteins in uterine fluid, will help identify novel markers of endometrial receptivity and also gain significant insights into the mechanisms underlying unexplained infertility, recurrent pregnancy losses, and other endometrial pathologies.

High-throughput Functional Genomics Identifies Regulators of Primary Human Beta Cell Proliferation

The expansion of cells for regenerative therapy will require the genetic dissection of complex regulatory mechanisms governing the proliferation of non-transformed human cells. Here, we report the development of a high-throughput RNAi screening strategy specifically for use in primary cells and demonstrate that silencing the cell cycle-dependent kinase inhibitors CDKN2C/p18 or CDKN1A/p21 facilitates cell cycle entry of quiescent adult human pancreatic beta cells. This work identifies p18 and p21 as novel targets for promoting proliferation of human beta cells and demonstrates the promise of functional genetic screens for dissecting therapeutically relevant state changes in primary human cells.

Cross-Species Integrative Functional Genomics in GeneWeaver Reveals a Role for Pafah1b1 in Altered Response to Alcohol.

Identifying the biological substrates of complex neurobehavioral traits such as alcohol dependency pose a tremendous challenge given the diverse model systems and phenotypic assessments used. To address this problem we have developed a platform for integrated analysis of high-throughput or genome-wide functional genomics studies. A wealth of such data exists, but it is often found in disparate, non-computable forms. Our interactive web-based software system, Gene Weaver (http://www.geneweaver.org), couples curated results from genomic studies to graph-theoretical tools for combinatorial analysis. Using this system we identified a gene underlying multiple alcohol-related phenotypes in four species. A search of over 60,000 gene sets in GeneWeaver's database revealed alcohol-related experimental results including genes identified in mouse genetic mapping studies, alcohol selected Drosophila lines, Rattus differential expression, and human alcoholic brains. We identified highly connected genes and compared these to genes currently annotated to alcohol-related behaviors and processes. The most highly connected gene not annotated to alcohol was Pafah1b1. Experimental validation using a Pafah1b1 conditional knock-out mouse confirmed that this gene is associated with an increased preference for alcohol and an altered thermoregulatory response to alcohol. Although this gene has not been previously implicated in alcohol-related behaviors, its function in various neural mechanisms makes a role in alcohol-related phenomena plausible. By making diverse cross-species functional genomics data readily computable, we were able to identify and confirm a novel alcohol-related gene that may have implications for alcohol use disorders and other effects of alcohol.

PGSB/MIPS Plant Genome Information Resources and Concepts for the Analysis of Complex Grass Genomes.

PGSB (Plant Genome and Systems Biology; formerly MIPS-Munich Institute for Protein Sequences) has been involved in developing, implementing and maintaining plant genome databases for more than a decade. Genome databases and analysis resources have focused on individual genomes and aim to provide flexible and maintainable datasets for model plant genomes as a backbone against which experimental data, e.g., from high-throughput functional genomics, can be organized and analyzed. In addition, genomes from both model and crop plants form a scaffold for comparative genomics, assisted by specialized tools such as the CrowsNest viewer to explore conserved gene order (synteny) between related species on macro- and micro-levels.The genomes of many economically important Triticeae plants such as wheat, barley, and rye present a great challenge for sequence assembly and bioinformatic analysis due to their enormous complexity and large genome size. Novel concepts and strategies have been developed to deal with these difficulties and have been applied to the genomes of wheat, barley, rye, and other cereals. This includes the GenomeZipper concept, reference-guided exome assembly, and "chromosome genomics" based on flow cytometry sorted chromosomes.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Genome-Wide RNAi Screening to Dissect the TGF-β Signal Transduction Pathway.

The transforming growth factor-β (TGF-β) family of cytokines figures prominently in regulation of embryonic development and adult tissue homeostasis from Drosophila to mammals. Genetic defects affecting TGF-β signaling underlie developmental disorders and diseases such as cancer in human. Therefore, delineating the molecular mechanism by which TGF-β regulates cell biology is critical for understanding normal biology and disease mechanisms. Forward genetic screens in model organisms and biochemical approaches in mammalian tissue culture were instrumental in initial characterization of the TGF-β signal transduction pathway. With complete sequence information of the genomes and the advent of RNA interference (RNAi) technology, genome-wide RNAi screening emerged as a powerful functional genomics approach to systematically delineate molecular components of signal transduction pathways. Here, we describe a protocol for image-based whole-genome RNAi screening aimed at identifying molecules required for TGF-β signaling into the nucleus. Using this protocol we examined >90 % of annotated Drosophila open reading frames (ORF) individually and successfully uncovered several novel factors serving critical roles in the TGF-β pathway. Thus cell-based high-throughput functional genomics can uncover new mechanistic insights on signaling pathways beyond what the classical genetics had revealed.

Genetic resources offer efficient tools for rice functional genomics research.

Rice is an important crop and major model plant for monocot functional genomics studies. With the establishment of various genetic resources for rice genomics, the next challenge is to systematically assign functions to predicted genes in the rice genome. Compared with the robustness of genome sequencing and bioinformatics techniques, progress in understanding the function of rice genes has lagged, hampering the utilization of rice genes for cereal crop improvement. The use of transfer DNA (T-DNA) insertional mutagenesis offers the advantage of uniform distribution throughout the rice genome, but preferentially in gene-rich regions, resulting in direct gene knockout or activation of genes within 20-30 kb up- and downstream of the T-DNA insertion site and high gene tagging efficiency. Here, we summarize the recent progress in functional genomics using the T-DNA-tagged rice mutant population. We also discuss important features of T-DNA activation- and knockout-tagging and promoter-trapping of the rice genome in relation to mutant and candidate gene characterizations and how to more efficiently utilize rice mutant populations and datasets for high-throughput functional genomics and phenomics studies by forward and reverse genetics approaches. These studies may facilitate the translation of rice functional genomics research to improvements of rice and other cereal crops.

CRISPR/Cas9 somatic multiplex-mutagenesis for high-throughput functional cancer genomics in mice

Here, we show CRISPR/Cas9-based targeted somatic multiplex-mutagenesis and its application for high-throughput analysis of gene function in mice. Using hepatic single guide RNA (sgRNA) delivery, we targeted large gene sets to induce hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). We observed Darwinian selection of target genes, which suppress tumorigenesis in the respective cellular/tissue context, such as Pten or Cdkn2a, and conversely found low frequency of Brca1/2 alterations, explaining mutational spectra in human ICC/HCC. Our studies show that multiplexed CRISPR/Cas9 can be used for recessive genetic screening or high-throughput cancer gene validation in mice. The analysis of CRISPR/Cas9-induced tumors provided support for a major role of chromatin modifiers in hepatobiliary tumorigenesis, including that of ARID family proteins, which have recently been reported to be mutated in ICC/HCC. We have also comprehensively characterized the frequency and size of chromosomal alterations induced by combinatorial sgRNA delivery and describe related limitations of CRISPR/Cas9 multiplexing, as well as opportunities for chromosome engineering in the context of hepatobiliary tumorigenesis. Our study describes novel approaches to model and study cancer in a high-throughput multiplexed format that will facilitate the functional annotation of cancer genomes.

12 Invited lecture: High-throughput functional genomics – An emerging field in head and neck cancer research

Introduction Head and neck squamous cell carcinomas (HNSCC) belong to the six most frequent tumors worldwide, and the incidence is rising. HNSCCs develop in the mucosal linings of the upper aerodigestive tract, and are preceded by prenoplastic changes, a phenomenon coined as “field cancerization”. Risk factors for HNSCC are smoking and excessive consumption of alcohol. Moreover, there are genetic predisposition syndromes, most particularly Fanconi anemia, associated with a very high risk for HNSCC. In recent years it became evident that also infection with the human papillomavirus (HPV) causes HNSCC, particularly tumors that arise in the oropharynx. HNSCC caused by HPV is a distinct class of tumors, both at the clinical and molecular level. Despite the application of combined treatment modalities that may severely impact quality of life with disfigurement, problems with speech and swallowing, the prognosis of HNSCC remains disappointing. New effective and less toxic treatments are urgently awaited. As a first step we tried to identify novel druggable gene targets. Material and methods Two tumor cell lines were transfected with a genome-wide siRNA library, and cell survival assayed. Sublibraries of lethal siRNAs were rescreened on a larger panel of HPV-positive and HPV-negative cell lines as well as primary fibroblasts. Data were normalized and compared by bioinformatics. Druggable gene targets were mined. Results We identified 362 genes that cause cell death in cancer cell lines, while only 20% of these cause cell death in primary fibroblasts. There were large differences between cell lines. One of the gene hits for which a small molecule inhibitor was available was analyzed in more detail. The inhibitor showed high efficacy in xenografted mouse models. Conclusion In head and neck cancer only few druggable oncogenic mutations occur. Nonetheless using functional genetic screens and genome editing methods multiple gene targets can be identified that might be exploited for improved treatment protocols.

Application of Functional Genomics for Bovine Respiratory Disease Diagnostics.

Bovine respiratory disease (BRD) is the most common economically important disease affecting cattle. For developing accurate diagnostics that can predict disease susceptibility/resistance and stratification, it is necessary to identify the molecular mechanisms that underlie BRD. To study the complex interactions among the bovine host and the multitude of viral and bacterial pathogens, as well as the environmental factors associated with BRD etiology, genome-scale high-throughput functional genomics methods such as microarrays, RNA-seq, and proteomics are helpful. In this review, we summarize the progress made in our understanding of BRD using functional genomics approaches. We also discuss some of the available bioinformatics resources for analyzing high-throughput data, in the context of biological pathways and molecular interactions. Although resources for studying host response to infection are avail-able, the corresponding information is lacking for majority of BRD pathogens, impeding progress in identifying diagnostic signatures for BRD using functional genomics approaches.

RNA-seq Transcriptional Profiling of Peripheral Blood Leukocytes from Cattle Infected with Mycobacterium bovis.

Bovine tuberculosis, caused by infection with Mycobacterium bovis, is a major endemic disease affecting cattle populations worldwide, despite the implementation of stringent surveillance and control programs in many countries. The development of high-throughput functional genomics technologies, including gene expression microarrays and RNA-sequencing (RNA-seq), has enabled detailed analysis of the host transcriptome to M. bovis infection, particularly at the macrophage and peripheral blood level. In the present study, we have analyzed the peripheral blood leukocyte (PBL) transcriptome of eight natural M. bovis-infected and eight age- and sex-matched non-infected control Holstein-Friesian animals using RNA-seq. In addition, we compared gene expression profiles generated using RNA-seq with those previously generated using the high-density Affymetrix® GeneChip® Bovine Genome Array platform from the same PBL-extracted RNA. A total of 3,250 differentially expressed (DE) annotated genes were detected in the M. bovis-infected samples relative to the controls (adjusted P-value ≤0.05), with the number of genes displaying decreased relative expression (1,671) exceeding those with increased relative expression (1,579). Ingenuity® Systems Pathway Analysis (IPA) of all DE genes revealed enrichment for genes with immune function. Notably, transcriptional suppression was observed among several of the top-ranking canonical pathways including Leukocyte Extravasation Signaling. Comparative platform analysis demonstrated that RNA-seq detected a larger number of annotated DE genes (3,250) relative to the microarray (1,398), of which 917 genes were common to both technologies and displayed the same direction of expression. Finally, we show that RNA-seq had an increased dynamic range compared to the microarray for estimating differential gene expression.

Genomics of wood-degrading fungi

Woody plants convert the energy of the sun into lignocellulosic biomass, which is an abundant substrate for bioenergy production. Fungi, especially wood decayers from the class Agaricomycetes, have evolved ways to degrade lignocellulose into its monomeric constituents, and understanding this process may facilitate the development of biofuels. Over the past decade genomics has become a powerful tool to study the Agaricomycetes. In 2004 the first sequenced genome of the white rot fungus Phanerochaete chrysosporium revealed a rich catalog of lignocellulolytic enzymes. In the decade that followed the number of genomes of Agaricomycetes grew to more than 75 and revealed a diversity of wood-decaying strategies. New technologies for high-throughput functional genomics are now needed to further study these organisms.

RNA and small molecule probes of hiPSC‐cardiomyocyte differentiation and physiology (207.1)

There is an urgent need for therapies that reverse the course of ventricular dysfunction in heart failure, which is a leading cause of morbidity and mortality. We have performed high throughput chemical and functional genomics screens probing cardiomyocyte differentiation and contractility, and used the results to construct protein‐protein interaction networks controlling these complex biological processes. To measure cardiac contractility in high throughput, we have used an automated microscopy platform (Vala Sciences) that optically measures heart muscle cell action potential and calcium transient kinetics and morphology. By screening static and kinetic endpoint assays against whole genome microRNA libraries, we have reconstructed microRNA:protein networks that control myocyte differentiation and contractility. This approach has revealed a number of microRNAs that direct differentiation and modulate physiological function, including microRNAs that adversely affect heart function in vivo. Targeting one of microRNAs with an antisense RNA is able to enhance echocardiographic and hemodynamic indices of heart function in vivo and halt the progression of established heart failure in a mouse pressure overload model.

A Systems Medicine Clinical Platform for Understanding and Managing Non- Communicable Diseases

Non-Communicable Diseases (NCDs) are among the most pressing global health problems of the twenty-first century. Their rising incidence and prevalence is linked to severe morbidity and mortality, and they are putting economic and managerial pressure on healthcare systems around the world. Moreover, NCDs are impeding healthy aging by negatively affecting the quality of life of a growing number of the global population. NCDs result from the interaction of various genetic, environmental and habitual factors, and cluster in complex ways, making the complex identification of resulting phenotypes not only difficult, but also a top research priority. The degree of complexity required to interpret large patient datasets generated by advanced high-throughput functional genomics assays has now increased to the point that novel computational biology approaches are essential to extract information that is relevant to the clinical decision-making process. Consequently, system-level models that interpret the interactions between extensive tissues, cellular and molecular measurements and clinical features are also being created to identify new disease phenotypes, so that disease definition and treatment are optimized, and novel therapeutic targets discovered. Likewise, Systems Medicine (SM) platforms applied to extensively-characterized patients provide a basis for more targeted clinical trials, and represent a promising tool to achieve better prevention and patient care, thereby promoting healthy aging globally. The present paper: (1) reviews the novel systems approaches to NCDs; (2) discusses how to move efficiently from Systems Biology to Systems Medicine; and (3) presents the scientific and clinical background of the San Raffaele Systems Medicine Platform.

TRANSGENIC ASIATIC GRAIN LEGUMES FOR SALT TOLERANCE AND FUNCTIONAL GENOMICS

The Asiatic Vigna species consists of several grain legumes that are native to tropical regions of new world. A number of them are of considerable economic importance in many developing countries. Abiotic stresses such as high salinity and low water availability are major causes of their yield losses and significantly affect their sustainable production. Mungbean (Vigna radiata) and cowpea (Vigna unguiculata) are two of the most important grain legumes of Vigna genus grown in increasingly hostile soil, with salinity and deprived water conditions both expected to worsen in future. Both mungbean and cowpea are classified as glycophytes (salt-sensitive). Exploitation of genetic variation of phenotypic traits through molecular breeding and plant translational genomics, that use knowledge and genes discovered in model plants, could facilitate improving tolerance in these two crops to salinity and drought. High-throughput functional genomics tools, such as transcriptomics, metabolomics, proteomics, and ionomics could be of immense use in investigating the molecular responses to salinity and drought in land races and wild germplasms carrying phenotypic variations for abiotic stress tolerance. Development of salt- and drought-tolerant mungbean and cowpea could play an important role in ensuring in dietary protein supply, improving human health and nutrition, and enhancing ecosystem resilience, especially in developing countries of Asia. In this article, we review and discuss the current knowledge of molecular basis of salt tolerance in model plants and the extent to which they can be implemented in Asiatic grain legumes for improvement to salinity.