Showing papers on "Gene published in 2016"
Broad Institute1, Harvard University2, Boston Children's Hospital3, University of Washington4, University of Arizona5, Cardiff University6, Google7, Icahn School of Medicine at Mount Sinai8, Samsung Medical Center9, Vertex Pharmaceuticals10, University of Michigan11, University of Cambridge12, State University of New York Upstate Medical University13, Karolinska Institutet14, University of Eastern Finland15, Wellcome Trust Centre for Human Genetics16, University of Oxford17, Cedars-Sinai Medical Center18, University of Ottawa19, University of Pennsylvania20, University of North Carolina at Chapel Hill21, University of Helsinki22, University of California, San Diego23, University of Mississippi Medical Center24
TL;DR: The aggregation and analysis of high-quality exome (protein-coding region) DNA sequence data for 60,706 individuals of diverse ancestries generated as part of the Exome Aggregation Consortium (ExAC) provides direct evidence for the presence of widespread mutational recurrence.
Abstract: Large-scale reference data sets of human genetic variation are critical for the medical and functional interpretation of DNA sequence changes. Here we describe the aggregation and analysis of high-quality exome (protein-coding region) DNA sequence data for 60,706 individuals of diverse ancestries generated as part of the Exome Aggregation Consortium (ExAC). This catalogue of human genetic diversity contains an average of one variant every eight bases of the exome, and provides direct evidence for the presence of widespread mutational recurrence. We have used this catalogue to calculate objective metrics of pathogenicity for sequence variants, and to identify genes subject to strong selection against various classes of mutation; identifying 3,230 genes with near-complete depletion of predicted protein-truncating variants, with 72% of these genes having no currently established human disease phenotype. Finally, we demonstrate that these data can be used for the efficient filtering of candidate disease-causing variants, and for the discovery of human 'knockout' variants in protein-coding genes.
8,758 citations
TL;DR: The KEGG GENES database now includes viruses, plasmids, and the addendum category for functionally characterized proteins that are not represented in complete genomes, and new automatic annotation servers, BlastKOalA and GhostKOALA, are made available utilizing the non-redundant pangenome data set generated from theGENES database.
Abstract: KEGG (http://www.kegg.jp/ or http://www.genome.jp/kegg/) is an integrated database resource for biological interpretation of genome sequences and other high-throughput data. Molecular functions of genes and proteins are associated with ortholog groups and stored in the KEGG Orthology (KO) database. The KEGG pathway maps, BRITE hierarchies and KEGG modules are developed as networks of KO nodes, representing high-level functions of the cell and the organism. Currently, more than 4000 complete genomes are annotated with KOs in the KEGG GENES database, which can be used as a reference data set for KO assignment and subsequent reconstruction of KEGG pathways and other molecular networks. As an annotation resource, the following improvements have been made. First, each KO record is re-examined and associated with protein sequence data used in experiments of functional characterization. Second, the GENES database now includes viruses, plasmids, and the addendum category for functionally characterized proteins that are not represented in complete genomes. Third, new automatic annotation servers, BlastKOALA and GhostKOALA, are made available utilizing the non-redundant pangenome data set generated from the GENES database. As a resource for translational bioinformatics, various data sets are created for antimicrobial resistance and drug interaction networks.
4,847 citations
TL;DR: By positioning histological sections on arrayed reverse transcription primers with unique positional barcodes, this work demonstrates high-quality RNA-sequencing data with maintained two-dimensional positional information from the mouse brain and human breast cancer.
Abstract: Analysis of the pattern of proteins or messengerRNAs (mRNAs) in histological tissue sections is a cornerstone in biomedical research and diagnostics. This typically involves the visualization of a few proteins or expressed genes at a time. We have devised a strategy, which we call “spatial transcriptomics,” that allows visualization and quantitative analysis of the transcriptome with spatial resolution in individual tissue sections. By positioning histological sections on arrayed reverse transcription primers with unique positional barcodes, we demonstrate high-quality RNA-sequencing data with maintained two-dimensional positional information from the mouse brain and human breast cancer. Spatial transcriptomics provides quantitative gene expression data and visualization of the distribution of mRNAs within tissue sections and enables novel types of bioinformatics analyses, valuable in research and diagnostics.
1,741 citations
Cambridge University Hospitals NHS Foundation Trust1, Wellcome Trust Sanger Institute2, Lund University3, Erasmus University Medical Center4, Radboud University Nijmegen5, European Bioinformatics Institute6, University of Oslo7, Oslo University Hospital8, Gachon University9, Netherlands Cancer Institute10, Université libre de Bruxelles11, University of Antwerp12, Harvard University13, University of Amsterdam14, University of Ulsan15, Hanyang University16, Memorial Sloan Kettering Cancer Center17, University of Texas MD Anderson Cancer Center18, French Institute of Health and Medical Research19, Ninewells Hospital20, ICM Partners21, University of Queensland22, University of Iceland23, Curie Institute24, University of Cambridge25, Institute of Cancer Research26, King's College London27, University of Bergen28, Singapore General Hospital29
TL;DR: This analysis of all classes of somatic mutation across exons, introns and intergenic regions highlights the repertoire of cancer genes and mutational processes operative, and progresses towards a comprehensive account of the somatic genetic basis of breast cancer.
Abstract: We analysed whole-genome sequences of 560 breast cancers to advance understanding of the driver mutations conferring clonal advantage and the mutational processes generating somatic mutations. We found that 93 protein-coding cancer genes carried probable driver mutations. Some non-coding regions exhibited high mutation frequencies, but most have distinctive structural features probably causing elevated mutation rates and do not contain driver mutations. Mutational signature analysis was extended to genome rearrangements and revealed twelve base substitution and six rearrangement signatures. Three rearrangement signatures, characterized by tandem duplications or deletions, appear associated with defective homologous-recombination-based DNA repair: one with deficient BRCA1 function, another with deficient BRCA1 or BRCA2 function, the cause of the third is unknown. This analysis of all classes of somatic mutation across exons, introns and intergenic regions highlights the repertoire of cancer genes and mutational processes operating, and progresses towards a comprehensive account of the somatic genetic basis of breast cancer.
1,696 citations
Harvard University1, Broad Institute2, University of California, Los Angeles3, VU University Medical Center4, VU University Amsterdam5, North Carolina State University6, Karolinska Institutet7, University of North Carolina at Chapel Hill8, University of Tampere9, Turku University Hospital10, University of Turku11, University of Eastern Finland12
TL;DR: A powerful strategy that integrates gene expression measurements with summary association statistics from large-scale genome-wide association studies (GWAS) to identify genes whose cis-regulated expression is associated with complex traits is introduced.
Abstract: Many genetic variants influence complex traits by modulating gene expression, thus altering the abundance of one or multiple proteins. Here we introduce a powerful strategy that integrates gene expression measurements with summary association statistics from large-scale genome-wide association studies (GWAS) to identify genes whose cis-regulated expression is associated with complex traits. We leverage expression imputation from genetic data to perform a transcriptome-wide association study (TWAS) to identify significant expression-trait associations. We applied our approaches to expression data from blood and adipose tissue measured in ∼ 3,000 individuals overall. We imputed gene expression into GWAS data from over 900,000 phenotype measurements to identify 69 new genes significantly associated with obesity-related traits (BMI, lipids and height). Many of these genes are associated with relevant phenotypes in the Hybrid Mouse Diversity Panel. Our results showcase the power of integrating genotype, gene expression and phenotype to gain insights into the genetic basis of complex traits.
1,473 citations
TL;DR: It is shown that it is possible to make hundreds of thousands permutations in a few minutes, which leads to very accurate p-values, which allows applying standard FDR correction procedures, which are more accurate than the ones currently used.
Abstract: Gene set enrichment analysis is a widely used tool for analyzing gene
expression data. However, current implementations are slow due to a large
number of required samples for the analysis to have a good statistical power.
In this paper we present a novel algorithm, that efficiently reuses
one sample multiple times and thus speeds up the analysis.
We show that it is possible to make hundreds of thousands permutations
in a few minutes, which leads to very accurate p-values. This, in turn,
allows applying standard FDR correction procedures, which are
more accurate than the ones currently used.
The method is implemented in a form of an R package and
is freely available at \url{https://github.com/ctlab/fgsea}.
1,221 citations
TL;DR: Analysis of transcriptomes of thousands of human islet cells from healthy and type 2 diabetic donors demonstrated the utility of the generated single-cell gene expression resource, and revealed subpopulations of α, β, and acinar cells.
1,098 citations
TL;DR: This work set out to define a minimal cellular genome experimentally by designing and building one, then testing it for viability, and applied whole-genome design and synthesis to the problem of minimizing a cellular genome.
Abstract: We used whole-genome design and complete chemical synthesis to minimize the 1079-kilobase pair synthetic genome of Mycoplasma mycoides JCVI-syn1.0. An initial design, based on collective knowledge of molecular biology combined with limited transposon mutagenesis data, failed to produce a viable cell. Improved transposon mutagenesis methods revealed a class of quasi-essential genes that are needed for robust growth, explaining the failure of our initial design. Three cycles of design, synthesis, and testing, with retention of quasi-essential genes, produced JCVI-syn3.0 (531 kilobase pairs, 473 genes), which has a genome smaller than that of any autonomously replicating cell found in nature. JCVI-syn3.0 retains almost all genes involved in the synthesis and processing of macromolecules. Unexpectedly, it also contains 149 genes with unknown biological functions. JCVI-syn3.0 is a versatile platform for investigating the core functions of life and for exploring whole-genome design.
1,047 citations
University of Toronto1, University of Minnesota2, Princeton University3, Tufts University4, European Bioinformatics Institute5, German Cancer Research Center6, Lunenfeld-Tanenbaum Research Institute7, Princess Margaret Cancer Centre8, University of Tokyo9, University College London10, Imperial College London11, Union University12, Huazhong University of Science and Technology13, Stanford University14
TL;DR: A global genetic interaction network highlights the functional organization of a cell and provides a resource for predicting gene and pathway function and how coherent sets of negative or positive genetic interactions connect protein complex and pathways to map a functional wiring diagram of the cell.
Abstract: INTRODUCTION Genetic interactions occur when mutations in two or more genes combine to generate an unexpected phenotype. An extreme negative or synthetic lethal genetic interaction occurs when two mutations, neither lethal individually, combine to cause cell death. Conversely, positive genetic interactions occur when two mutations produce a phenotype that is less severe than expected. Genetic interactions identify functional relationships between genes and can be harnessed for biological discovery and therapeutic target identification. They may also explain a considerable component of the undiscovered genetics associated with human diseases. Here, we describe construction and analysis of a comprehensive genetic interaction network for a eukaryotic cell. RATIONALE Genome sequencing projects are providing an unprecedented view of genetic variation. However, our ability to interpret genetic information to predict inherited phenotypes remains limited, in large part due to the extensive buffering of genomes, making most individual eukaryotic genes dispensable for life. To explore the extent to which genetic interactions reveal cellular function and contribute to complex phenotypes, and to discover the general principles of genetic networks, we used automated yeast genetics to construct a global genetic interaction network. RESULTS We tested most of the ~6000 genes in the yeast Saccharomyces cerevisiae for all possible pairwise genetic interactions, identifying nearly 1 million interactions, including ~550,000 negative and ~350,000 positive interactions, spanning ~90% of all yeast genes. Essential genes were network hubs, displaying five times as many interactions as nonessential genes. The set of genetic interactions or the genetic interaction profile for a gene provides a quantitative measure of function, and a global network based on genetic interaction profile similarity revealed a hierarchy of modules reflecting the functional architecture of a cell. Negative interactions connected functionally related genes, mapped core bioprocesses, and identified pleiotropic genes, whereas positive interactions often mapped general regulatory connections associated with defects in cell cycle progression or cellular proteostasis. Importantly, the global network illustrates how coherent sets of negative or positive genetic interactions connect protein complex and pathways to map a functional wiring diagram of the cell. CONCLUSION A global genetic interaction network highlights the functional organization of a cell and provides a resource for predicting gene and pathway function. This network emphasizes the prevalence of genetic interactions and their potential to compound phenotypes associated with single mutations. Negative genetic interactions tend to connect functionally related genes and thus may be predicted using alternative functional information. Although less functionally informative, positive interactions may provide insights into general mechanisms of genetic suppression or resiliency. We anticipate that the ordered topology of the global genetic network, in which genetic interactions connect coherently within and between protein complexes and pathways, may be exploited to decipher genotype-to-phenotype relationships.
1,037 citations
TL;DR: Perturb-seq as discussed by the authors combines single-cell RNA sequencing and clustered regularly interspaced short palindromic repeats (CRISPR)-based perturbations to perform many such assays in a pool.
1,019 citations
TL;DR: Nucleosome spacing inferred from cfDNA in healthy individuals correlates most strongly with epigenetic features of lymphoid and myeloid cells, consistent with hematopoietic cell death as the normal source of cfDNA.
TL;DR: Population modeling and cage experiments indicate that a CRISPR-Cas9 construct targeting one of these loci meets the minimum requirement for a gene drive targeting female reproduction in an insect population, which could expedite the development of gene drives to suppress mosquito populations to levels that do not support malaria transmission.
Abstract: Gene drive systems that enable super-Mendelian inheritance of a transgene have the potential to modify insect populations over a timeframe of a few years. We describe CRISPR-Cas9 endonuclease constructs that function as gene drive systems in Anopheles gambiae, the main vector for malaria. We identified three genes (AGAP005958, AGAP011377 and AGAP007280) that confer a recessive female-sterility phenotype upon disruption, and inserted into each locus CRISPR-Cas9 gene drive constructs designed to target and edit each gene. For each targeted locus we observed a strong gene drive at the molecular level, with transmission rates to progeny of 91.4 to 99.6%. Population modeling and cage experiments indicate that a CRISPR-Cas9 construct targeting one of these loci, AGAP007280, meets the minimum requirement for a gene drive targeting female reproduction in an insect population. These findings could expedite the development of gene drives to suppress mosquito populations to levels that do not support malaria transmission.
TL;DR: Cross-talk among neighbouring genes is a prevalent phenomenon that can involve multiple mechanisms and cis-regulatory signals, including a role for RNA splice sites, and mechanisms may explain the function and evolution of some genomic loci that produce lncRNAs and broadly contribute to the regulation of both coding and non-coding genes.
Abstract: Mammalian genomes are pervasively transcribed to produce thousands of long non-coding RNAs (lncRNAs) A few of these lncRNAs have been shown to recruit regulatory complexes through RNA-protein interactions to influence the expression of nearby genes, and it has been suggested that many other lncRNAs can also act as local regulators Such local functions could explain the observation that lncRNA expression is often correlated with the expression of nearby genes However, these correlations have been challenging to dissect and could alternatively result from processes that are not mediated by the lncRNA transcripts themselves For example, some gene promoters have been proposed to have dual functions as enhancers, and the process of transcription itself may contribute to gene regulation by recruiting activating factors or remodelling nucleosomes Here we use genetic manipulation in mouse cell lines to dissect 12 genomic loci that produce lncRNAs and find that 5 of these loci influence the expression of a neighbouring gene in cis Notably, none of these effects requires the specific lncRNA transcripts themselves and instead involves general processes associated with their production, including enhancer-like activity of gene promoters, the process of transcription, and the splicing of the transcript Furthermore, such effects are not limited to lncRNA loci: we find that four out of six protein-coding loci also influence the expression of a neighbour These results demonstrate that cross-talk among neighbouring genes is a prevalent phenomenon that can involve multiple mechanisms and cis-regulatory signals, including a role for RNA splice sites These mechanisms may explain the function and evolution of some genomic loci that produce lncRNAs and broadly contribute to the regulation of both coding and non-coding genes
Icahn School of Medicine at Mount Sinai1, Sage Bionetworks2, Duke University3, University of Pittsburgh4, National Institutes of Health5, Carnegie Mellon University6, High Point University7, Cedars-Sinai Medical Center8, Broad Institute9, Rush University Medical Center10, Brigham and Women's Hospital11, Karolinska Institutet12, University of North Carolina at Chapel Hill13, Takeda Pharmaceutical Company14, University of Pennsylvania15, University of Trento16
TL;DR: It is shown that schizophrenia is polygenic and the utility of this resource of gene expression and its genetic regulation for mechanistic interpretations of genetic liability for brain diseases is highlighted.
Abstract: Over 100 genetic loci harbor schizophrenia associated variants, yet how these variants confer liability is uncertain. The CommonMind Consortium sequenced RNA from dorsolateral prefrontal cortex of schizophrenia cases (N = 258) and control subjects (N = 279), creating a resource of gene expression and its genetic regulation. Using this resource, ~20% of schizophrenia loci have variants that could contribute to altered gene expression and liability. In five loci, only a single gene was involved: FURIN, TSNARE1, CNTN4, CLCN3, or SNAP91. Altering expression of FURIN, TSNARE1, or CNTN4 changes neurodevelopment in zebrafish; knockdown of FURIN in human neural progenitor cells yields abnormal migration. Of 693 genes showing significant case/control differential expression, their fold changes are ≤ 1.33, and an independent cohort yields similar results. Gene co-expression implicates a network relevant for schizophrenia. Our findings show schizophrenia is polygenic and highlight the utility of this resource for mechanistic interpretations of genetic liability for brain diseases.
TL;DR: An automated platform is developed that uses FACS, robotics, and the CEL-Seq2 protocol to obtain the transcriptomes of thousands of single pancreatic cells from deceased organ donors, allowing in silico purification of all main pancreatic cell types.
Abstract: To understand organ function, it is important to have an inventory of its cell types and of their corresponding marker genes. This is a particularly challenging task for human tissues like the pancreas, because reliable markers are limited. Hence, transcriptome-wide studies are typically done on pooled islets of Langerhans, obscuring contributions from rare cell types and of potential subpopulations. To overcome this challenge, we developed an automated platform that uses FACS, robotics, and the CEL-Seq2 protocol to obtain the transcriptomes of thousands of single pancreatic cells from deceased organ donors, allowing in silico purification of all main pancreatic cell types. We identify cell type-specific transcription factors and a subpopulation of REG3A-positive acinar cells. We also show that CD24 and TM4SF4 expression can be used to sort live alpha and beta cells with high purity. This resource will be useful for developing a deeper understanding of pancreatic biology and pathophysiology of diabetes mellitus.
TL;DR: A comprehensive transcriptional map of human embryo development, including the sequenced transcriptomes of 1,529 individual cells from 88 human preimplantation embryos, shows that cells undergo an intermediate state of co-expression of lineage-specific genes, followed by a concurrent establishment of the trophectoderm, epiblast, and primitive endoderm lineages, which coincide with blastocyst formation.
TL;DR: This work uses promoter capture Hi-C to identify interacting regions of 31,253 promoters in 17 human primary hematopoietic cell types and shows that promoter interactions are highly cell type specific and enriched for links between active promoters and epigenetically marked enhancers.
University of California, Berkeley1, United States Department of Energy2, Nagoya University3, University of Texas at Austin4, Ulsan National Institute of Science and Technology5, National Institute of Genetics6, Hiroshima University7, Hokkaido University8, University of Tokyo9, Radboud University Nijmegen10, Salk Institute for Biological Studies11, Nagahama Institute of Bio-Science and Technology12, Yamagata University13, Okinawa Institute of Science and Technology14, Tokyo Institute of Technology15, University of Tokushima16, Harry Perkins Institute of Medical Research17, Rikkyo University18, University of Maryland, Baltimore19, Kitasato University20, University of Chicago21, National Institute of Advanced Industrial Science and Technology22, National Institutes of Natural Sciences, Japan23, Illumina24, University of Washington25, University of Virginia26, Niigata University27, University of Rochester28, Cincinnati Children's Hospital Medical Center29, University of Calgary30, University of Iowa31, University of Basel32, Graduate University for Advanced Studies33, National Institute of Informatics34
TL;DR: The Xenopus laevis genome is sequenced and it is estimated that the two diploid progenitor species diverged around 34 million years ago and combined to form an allotetraploid around 17–18 Ma, where more than 56% of all genes were retained in two homoeologous copies.
Abstract: To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of 'fossil' transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17-18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.
TL;DR: Recent advances in understanding the physiological function of the TET proteins and their role in regulating DNA methylation and transcription are highlighted and some of the key outstanding questions are discussed.
Abstract: The pattern of DNA methylation at cytosine bases in the genome is tightly linked to gene expression, and DNA methylation abnormalities are often observed in diseases. The ten eleven translocation (TET) enzymes oxidize 5-methylcytosines (5mCs) and promote locus-specific reversal of DNA methylation. TET genes, and especially TET2, are frequently mutated in various cancers, but how the TET proteins contribute to prevent the onset and maintenance of these malignancies is largely unknown. Here, we highlight recent advances in understanding the physiological function of the TET proteins and their role in regulating DNA methylation and transcription. In addition, we discuss some of the key outstanding questions in the field.
TL;DR: It is found that ERVs have shaped the evolution of a transcriptional network underlying the interferon (IFN) response, a major branch of innate immunity, and that lineage-specific ERV have dispersed numerous IFN-inducible enhancers independently in diverse mammalian genomes.
Abstract: Endogenous retroviruses (ERVs) are abundant in mammalian genomes and contain sequences modulating transcription. The impact of ERV propagation on the evolution of gene regulation remains poorly understood. We found that ERVs have shaped the evolution of a transcriptional network underlying the interferon (IFN) response, a major branch of innate immunity, and that lineage-specific ERVs have dispersed numerous IFN-inducible enhancers independently in diverse mammalian genomes. CRISPR-Cas9 deletion of a subset of these ERV elements in the human genome impaired expression of adjacent IFN-induced genes and revealed their involvement in the regulation of essential immune functions, including activation of the AIM2 inflammasome. Although these regulatory sequences likely arose in ancient viruses, they now constitute a dynamic reservoir of IFN-inducible enhancers fueling genetic innovation in mammalian immune defenses.
TL;DR: This transient expression-based genome-editing system is highly efficient and specific for producing transgene-free and homozygous wheat mutants in the T0 generation, and may be applicable to other plant species.
Abstract: Editing plant genomes is technically challenging in hard-to-transform plants and usually involves transgenic intermediates, which causes regulatory concerns. Here we report two simple and efficient genome-editing methods in which plants are regenerated from callus cells transiently expressing CRISPR/Cas9 introduced as DNA or RNA. This transient expression-based genome-editing system is highly efficient and specific for producing transgene-free and homozygous wheat mutants in the T0 generation. We demonstrate our protocol to edit genes in hexaploid bread wheat and tetraploid durum wheat, and show that we are able to generate mutants with no detectable transgenes. Our methods may be applicable to other plant species, thus offering the potential to accelerate basic and applied plant genome-engineering research.
TL;DR: The accessible chromatin landscape in exhausted CD8+ T cells is defined and shown that it is distinct from functional memory CD8 + T cells, suggesting that exhausted T cells are a distinct lineage.
Abstract: Exhausted T cells in cancer and chronic viral infection express distinctive patterns of genes, including sustained expression of programmed cell death protein 1 (PD-1) However, the regulation of gene expression in exhausted T cells is poorly understood Here, we define the accessible chromatin landscape in exhausted CD8+ T cells and show that it is distinct from functional memory CD8+ T cells Exhausted CD8+ T cells in humans and a mouse model of chronic viral infection acquire a state-specific epigenetic landscape organized into functional modules of enhancers Genome editing shows that PD-1 expression is regulated in part by an exhaustion-specific enhancer that contains essential RAR, T-bet, and Sox3 motifs Functional enhancer maps may offer targets for genome editing that alter gene expression preferentially in exhausted CD8+ T cells
TL;DR: This Primer outlines the discovery, roles and regulation of circular RNAs, focussing on their potential functions during development and on the regulation of and functional roles played by these molecules.
Abstract: Just a few years ago, it had been assumed that the dominant RNA isoforms produced from eukaryotic genes were variants of messenger RNA, functioning as intermediates in gene expression. In early 2012, however, a surprising discovery was made: circular RNA (circRNA) was shown to be a transcriptional product in thousands of human and mouse genes and in hundreds of cases constituted the dominant RNA isoform. Subsequent studies revealed that the expression of circRNAs is developmentally regulated, tissue and cell-type specific, and shared across the eukaryotic tree of life. These features suggest important functions for these molecules. Here, we describe major advances in the field of circRNA biology, focusing on the regulation of and functional roles played by these molecules.
TL;DR: Prevalence of mcr-2 in porcine colistin-resistant E. coli (11/53) in Belgium was higher than that of mCr-1 (7/53), and data call for an immediate introduction of mCR-2 screening in ongoing molecular epidemiological surveillance of colistIn-resistant Gram-negative pathogens.
Abstract: We identified a novel plasmid-mediated colistin-resistance gene in porcine and bovine colistin-resistant Escherichia coli that did not contain mcr-1. The gene, termed mcr-2, a 1,617 bp phosphoethanolamine transferase harboured on an IncX4 plasmid, has 76.7% nucleotide identity to mcr-1. Prevalence of mcr-2 in porcine colistin-resistant E. coli (11/53) in Belgium was higher than that of mcr-1 (7/53). These data call for an immediate introduction of mcr-2 screening in ongoing molecular epidemiological surveillance of colistin-resistant Gram-negative pathogens.
TL;DR: For the first time, virus resistance has been developed in cucumber, non-transgenically, not visibly affecting plant development and without long-term backcrossing, via a new technology that can be expected to be applicable to a wide range of crop plants.
Abstract: Genome editing in plants has been boosted tremendously by the development of CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) technology. This powerful tool allows substantial improvement in plant traits in addition to those provided by classical breeding. Here, we demonstrate the development of virus resistance in cucumber (Cucumis sativus L.) using Cas9/subgenomic RNA (sgRNA) technology to disrupt the function of the recessive eIF4E (eukaryotic translation initiation factor 4E) gene. Cas9/sgRNA constructs were targeted to the N' and C' termini of the eIF4E gene. Small deletions and single nucleotide polymorphisms (SNPs) were observed in the eIF4E gene targeted sites of transformed T1 generation cucumber plants, but not in putative off-target sites. Non-transgenic heterozygous eif4e mutant plants were selected for the production of non-transgenic homozygous T3 generation plants. Homozygous T3 progeny following Cas9/sgRNA that had been targeted to both eif4e sites exhibited immunity to Cucumber vein yellowing virus (Ipomovirus) infection and resistance to the potyviruses Zucchini yellow mosaic virus and Papaya ring spot mosaic virus-W. In contrast, heterozygous mutant and non-mutant plants were highly susceptible to these viruses. For the first time, virus resistance has been developed in cucumber, non-transgenically, not visibly affecting plant development and without long-term backcrossing, via a new technology that can be expected to be applicable to a wide range of crop plants.
TL;DR: Biolistic delivery of pre-assembled Cas9–gRNA ribonucleoproteins into maize embryo cells and regeneration of plants with both mutated and edited alleles are reported, opening new opportunities to accelerate breeding practices in a wide variety of crop species.
Abstract: Targeted DNA double-strand breaks have been shown to significantly increase the frequency and precision of genome editing. In the past two decades, several double-strand break technologies have been developed. CRISPR-Cas9 has quickly become the technology of choice for genome editing due to its simplicity, efficiency and versatility. Currently, genome editing in plants primarily relies on delivering double-strand break reagents in the form of DNA vectors. Here we report biolistic delivery of pre-assembled Cas9-gRNA ribonucleoproteins into maize embryo cells and regeneration of plants with both mutated and edited alleles. Using this method of delivery, we also demonstrate DNA- and selectable marker-free gene mutagenesis in maize and recovery of plants with mutated alleles at high frequencies. These results open new opportunities to accelerate breeding practices in a wide variety of crop species.
TL;DR: Together, these features raise fundamental questions regarding the regulation of circRNA in cis and in trans, and its function, which are enriched in the brain and increase in abundance during fetal development.
TL;DR: It is shown by chromosome conformation capture that genomic duplications in patient cells and genetically modified mice can result in the formation of new chromatin domains (neo-TADs) and that this process determines their molecular pathology.
Abstract: Chromosome conformation capture methods have identified subchromosomal structures of higher-order chromatin interactions called topologically associated domains (TADs) that are separated from each other by boundary regions. By subdividing the genome into discrete regulatory units, TADs restrict the contacts that enhancers establish with their target genes. However, the mechanisms that underlie partitioning of the genome into TADs remain poorly understood. Here we show by chromosome conformation capture (capture Hi-C and 4C-seq methods) that genomic duplications in patient cells and genetically modified mice can result in the formation of new chromatin domains (neo-TADs) and that this process determines their molecular pathology. Duplications of non-coding DNA within the mouse Sox9 TAD (intra-TAD) that cause female to male sex reversal in humans, showed increased contact of the duplicated regions within the TAD, but no change in the overall TAD structure. In contrast, overlapping duplications that extended over the next boundary into the neighbouring TAD (inter-TAD), resulted in the formation of a new chromatin domain (neo-TAD) that was isolated from the rest of the genome. As a consequence of this insulation, inter-TAD duplications had no phenotypic effect. However, incorporation of the next flanking gene, Kcnj2, in the neo-TAD resulted in ectopic contacts of Kcnj2 with the duplicated part of the Sox9 regulatory region, consecutive misexpression of Kcnj2, and a limb malformation phenotype. Our findings provide evidence that TADs are genomic regulatory units with a high degree of internal stability that can be sculptured by structural genomic variations. This process is important for the interpretation of copy number variations, as these variations are routinely detected in diagnostic tests for genetic disease and cancer. This finding also has relevance in an evolutionary setting because copy-number differences are thought to have a crucial role in the evolution of genome complexity.
TL;DR: It is suggested that the combination of disease resistance genes with other practices for pathogen control (pesticides, farming practices) may be a relevant management strategy to slow down the evolution of virulent pathogen genotypes.
Abstract: The efficacy of disease resistance genes in plants decreases over time because of the selection of virulent pathogen genotypes. A key goal of crop protection programs is to increase the durability of the resistance conferred by these genes. The spatial and temporal deployment of plant disease resistance genes is considered to be a major factor determining their durability. In the literature, four principal strategies combining resistance genes over time and space have been considered to delay the evolution of virulent pathogen genotypes: cultivars mixture, rotation, landscape deployment, pyramiding. We reviewed this literature with the aim of determining which deployment strategy results in the greatest durability of resistance genes. Although theoretical and empirical studies comparing deployment strategies of more than one resistance gene are very scarce, they suggest that the overall durability of disease resistance genes can be increased by combining their presence in the same plant (pyramiding). Retrospective analyses of field monitoring data also suggest that the pyramiding of disease resistance genes within a plant is the most durable strategy. By extension, we suggest that the combination of disease resistance genes with other practices for pathogen control (pesticides, farming practices) may be a relevant management strategy to slow down the evolution of virulent pathogen genotypes.
TL;DR: The results indicate that gene modification via CRISPR/Cas9 is a useful approach for enhancing blast resistance in rice and targeted multiple sites within OsERF922 by using Cas9/Multi-target-sgRNAs to obtain plants harboring mutations at two or three sites.
Abstract: Rice blast is one of the most destructive diseases affecting rice worldwide. The adoption of host resistance has proven to be the most economical and effective approach to control rice blast. In recent years, sequence-specific nucleases (SSNs) have been demonstrated to be powerful tools for the improvement of crops via gene-specific genome editing, and CRISPR/Cas9 is thought to be the most effective SSN. Here, we report the improvement of rice blast resistance by engineering a CRISPR/Cas9 SSN (C-ERF922) targeting the OsERF922 gene in rice. Twenty-one C-ERF922-induced mutant plants (42.0%) were identified from 50 T0 transgenic plants. Sanger sequencing revealed that these plants harbored various insertion or deletion (InDel) mutations at the target site. We showed that all of the C-ERF922-induced allele mutations were transmitted to subsequent generations. Mutant plants harboring the desired gene modification but not containing the transferred DNA were obtained by segregation in the T1 and T2 generations. Six T2 homozygous mutant lines were further examined for a blast resistance phenotype and agronomic traits, such as plant height, flag leaf length and width, number of productive panicles, panicle length, number of grains per panicle, seed setting percentage and thousand seed weight. The results revealed that the number of blast lesions formed following pathogen infection was significantly decreased in all 6 mutant lines compared with wild-type plants at both the seedling and tillering stages. Furthermore, there were no significant differences between any of the 6 T2 mutant lines and the wild-type plants with regard to the agronomic traits tested. We also simultaneously targeted multiple sites within OsERF922 by using Cas9/Multi-target-sgRNAs (C-ERF922S1S2 and C-ERF922S1S2S3) to obtain plants harboring mutations at two or three sites. Our results indicate that gene modification via CRISPR/Cas9 is a useful approach for enhancing blast resistance in rice.