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New Breeding Techniques: Detection and Identification of the Techniques and Derived Products
Abstract
Since the commercial releases of GMOs in the 90s, new genetic modification tools known as New breeding techniques have been developed for e.g. gene silencing or more precise genomic modifications such as Crispr-endonuclease based systems. As for GMOs several consumers view may prevail about the societal interest in agricultural production and food of such genetic modification. Ensuring the freedom of choice to consumers needs to develop detection tools which could infer the NBT nature of the modification technique used. This article reviews all the elements which could allow the identification and detection of such techniques and products.
... These related techniques include (i) protoplastisation 1 GMO Risk Assessment Agency 2 Oligonucleotide directed Mutagenesis 3 Interfering RNA to obtain isolated cells,(ii) in vitro culture, (iii) delivery of reagents (DNA, RNA, or RNA-associated or non-RNA-associated proteins), (iv) the selection procedure of modifying cells, (v) the elimination of these same selection markers for example by the Cre-Lox recombinase system, (vi) the callus differentiation, and (vii) the regeneration of seedlings before (viii) the habituation to culture conditions. All these mutations and epimutations follow a dynamic that has not been studied in detail until now (Bertheau, 2019;Filipecki and Malepszy, 2006;Lee and Seo, 2018;Mendizabal et al., 2014). However, these old-chapped related techniques still constitute a bottleneck in large-scale technique applications that have not been mastered, as recently noted (Ledford, 2016). ...
... The delivery of reagents, whether DNA, RNA (messenger or not) or proteins (such as RNA-associated nucleases for CRISPR-Cas RNPs 5 contaminated by foreign DNA of the multiplication organism DNA despite purification steps), is a highly empirical technical phase that induces genomic and epigenomic scars (Bertheau, 2019;Khalil, 2020;Ledford, 2016). ...
... Transgenesis and genome and epigenome editing methods share these related techniques and their unintended effects. The scars left after the excision of selection markers from modified cells is only one example (Ates et al., 2020;Bertheau, 2019;Jansing et al., 2019;Manimaran et al., 2011;Marx, 2015;Miguel and Marum, 2011;Ruffoni and Savona, 2013;Scahill et al., 2008;Volkova et al., 2020;Yau and Stewart, 2013). Therefore, most of the authors consider it necessary for the NBT to avoid all these in vitro culture phases, but the suggested options have not proven themselves (Hamada et al., 2017;Maher et al., 2020). ...
In 2018 the Court of Justice of the European Union recalled that organisms with genomes modified by artifactual techniques should be considered GMOs under European regulations. GMOs derived from cultures of cells isolated in vitro or from new genomic techniques must therefore be traceable. This chapter reviews the various technical steps and characteristics of those techniques causing genomic and epigenomic scars and signatures. These intentional and unintentional traces, some of which are already used for varietal identification, and are being standardized, can be used to identify these GMOs and differentiate them from natural mutants. The chapter suggests a routine procedure for operators and control laboratories to achieve this without additional costs.
... aflatoxin) or pathogens, GMOs are a highly political-technicalscientific issue. Thus, when they are discussed or explored in contemporary literature and media, the state-of-the-art techniques and scientific knowledge involved are only partially taken into account (Bertheau, 2019;Bertheau, 2007, 2009). Despite the passage of 30 years, the definition of GMO is still imprecise and contested. ...
... The use of different targets and signal amplification of variants, with or without hybridisation, makes available a range of qualitative and quantitative methods (QRT-PCR, LAMP, NASBA, LCR, SNPLex…) that can be chosen, depending on the target. These methodological approaches can be applied from the field to the laboratory, from point mutation to the junction sequence between rearranged elements and from single to multiple targets Bertheau, 2019;Chhalliyil et al., 2020). Associated with various decision support tools, they allow, via the matrix approach, the identification of any GMO. ...
... aflatoxin) or pathogens, GMOs are a highly political-technicalscientific issue. Thus, when they are discussed or explored in contemporary literature and media, the state-of-the-art techniques and scientific knowledge involved are only partially taken into account (Bertheau, 2019;Bertheau, 2007, 2009). Despite the passage of 30 years, the definition of GMO is still imprecise and contested. ...
... The use of different targets and signal amplification of variants, with or without hybridisation, makes available a range of qualitative and quantitative methods (QRT-PCR, LAMP, NASBA, LCR, SNPLex…) that can be chosen, depending on the target. These methodological approaches can be applied from the field to the laboratory, from point mutation to the junction sequence between rearranged elements and from single to multiple targets Bertheau, 2019;Chhalliyil et al., 2020). Associated with various decision support tools, they allow, via the matrix approach, the identification of any GMO. ...
Transgenic GMOs were welcomed in the 1990s due to the difficulties distinguishing genetic and epigenetic modifications from random mutagenesis and their ability to insert new nucleic sequences more rapidly but still randomly. Their marketing in Europe has been accompanied by health and environmental risk assessments, specific monitoring and traceability procedures to preserve the free choice of consumers and allow the coexistence of different supply chains. This chapter reviews the regulations, detection techniques, strategies and standards that have been put in place in the European Union since 1996 to ensure the analytical traceability of these GMOs. The capacity of the matrix approach, initially targeted at transgenic GMOs, to trace other types of GMOs is discussed in an accompanying chapter.
... Although the precision of genome editing and range of off-target and other unintended effects, especially in comparison with random mutagenesis, have been one focus of the discussion regarding regulatory status of genome-edited organisms [3,19,[22][23][24][25], a second, prominent focus has been feasibility of developing methods for detecting genome-edited organisms [3,7,10,[25][26][27][28][29]. One of the arguments put forward to justify regulating genome-edited crops differently from recombinant DNA-based GMOs is that Directive 2001/18/EC requires analysis-based surveillance of GMOs, while, it is claimed, there are many technical and regulatory challenges that make development of GMO regulation-compliant analytical identification and quantitation methods for genome-edited organisms difficult or even impossible [7,10,25,26,28,29]. ...
... One of the arguments put forward to justify regulating genome-edited crops differently from recombinant DNA-based GMOs is that Directive 2001/18/EC requires analysis-based surveillance of GMOs, while, it is claimed, there are many technical and regulatory challenges that make development of GMO regulation-compliant analytical identification and quantitation methods for genome-edited organisms difficult or even impossible [7,10,25,26,28,29]. However, these claims are controversial [3,27]. ...
Discussion regarding the regulatory status of genome-edited crops has focused on precision of editing and on doubts regarding the feasibility of analytical monitoring compliant with existing GMO regulations. Effective detection methods are important, both for regulatory enforcement and traceability in case of biosafety, environmental or socio-economic impacts. Here, we approach the analysis question for the first time in the laboratory and report the successful development of a quantitative PCR detection method for the first commercialized genome-edited crop, a canola with a single base pair edit conferring herbicide tolerance. The method is highly sensitive and specific (quantification limit, 0.05%), compatible with the standards of practice, equipment and expertise typical in GMO laboratories, and readily integrable into their analytical workflows, including use of the matrix approach. The method, validated by an independent laboratory, meets all legal requirements for GMO analytical methods in jurisdictions such as the EU, is consistent with ISO17025 accreditation standards and has been placed in the public domain. Having developed a qPCR method for the most challenging class of genome edits, single-nucleotide variants, this research suggests that qPCR-based method development may be applicable to virtually any genome-edited organism. This advance resolves doubts regarding the feasibility of extending the regulatory approach currently employed for recombinant DNA-based GMOs to genome-edited organisms.
... As a result, new detection approaches must be developed and evaluated for the detection of these small modifications in pure and mixed products, and to distinguish edited from non-edited organisms. Some theoretical papers discuss possible methodologies for the detection of NGT-GE organisms (Bertheau et al., 2019;Grohmann et al., 2019). ...
In this scientific advisory report, which offers guidance to public health policy-makers, the Superior Health Council of Belgium provides its opinion on the introduction of plants edited by cisgenesis or targeted mutagenesis using NGTs. This report aims at the same time to provide policy-makers with some specific recommendations on the proposal of the European Commission.
... The methodological detection of most other genome-edited products, however, still remains a major hurdle, especially considering that a genome-edited organism may have multiple targeted point mutations, rendering detection of multiple events particularly challenging. However, evidence has been compiled in a number of publications that could enable the detection of genomeedited products or even identify the techniques behind their development (Bertheau, 2019;Ribarits et al., 2021a;Ribarits et al., 2021b;Ribarits et al., 2022;Fraiture et al., 2022;Potthof et al., 2023). In addition, initiatives such as the Norwegian Foodprint program NORCE (https://foodprintproject.org/; accessed on 21 March 2023) aim to develop gene-editing detection tools for traceability and labelling of genetically modified (GM) products throughout the food chain. ...
Novel techniques such as CRISPR/Cas are increasingly being applied for the development of modern crops. However, the regulatory framework for production, labelling and handling of genome-edited organisms varies worldwide. Currently, the European Commission is raising the question whether genome-edited organisms should still be regulated as genetically modified organisms in the future or whether a deregulation should be implemented. In our paper, based on the outcome of a 2-year case study on oilseed rape in Austria, we show that seed spillage during import and subsequent transport and handling activities is a key factor for the unintended dispersal of seeds into the environment, the subsequent emergence of feral oilseed rape populations and their establishment and long-term persistence in natural habitats. These facts must likewise be considered in case of genome-edited oilseed rape contaminants that might be accidentally introduced with conventional kernels. We provide evidence that in Austria a high diversity of oilseed rape genotypes, including some with alleles not known from cultivated oilseed rape in Austria, exists at sites with high seed spillage and low weed management, rendering these sites of primary concern with respect to possible escape of genome-edited oilseed rape varieties into the environment. Since appropriate detection methods for single genome-edited oilseed rape events have only recently started to be successfully developed and the adverse effects of these artificial punctate DNA exchanges remain largely unknown, tracing the transmission and spread of these genetic modifications places high requirements on their monitoring, identification, and traceability.
... Zellkulturen sind in der Pflanzenzüchtung weit verbreitet, und Methoden zur Protoplasten-und Zellregeneration sind in der Pflanzenzüchtung oft unverzichtbar. Diese in vitro-Phasen können zu unerwünschten Effekten im Pflanzengenom führen, die als möglicher Ansatz für den Nachweis von NGT diskutiert wurden (Bertheau 2019). ...
In the EU, genetically modified organisms (GMOs) are subject to the authorization require-ments of Directive 2001/18/EC or Regulation (EC) No. 1829/2003. Application for authoriza-tion of a genetically modified plant requires description of identification and detection meth-ods. Those methods are used by control laboratories of the Member States to detect and identify genetically modified plants and to quantify their occurrence in food and feed. In its ruling of July 25, 2018 (C-528/16), the European Court of Justice determined that plants pro-duced with directed mutagenesis (genome editing) are covered by the regulations under Di-rective 2001/18/EC on the release and placing on the market of GMOs. For control laborato-ries this poses specific challenges for detection, identification and quantification of genome-edited plants.
Genome editing techniques allow targeted modifications at predefined sites in the genome of organisms. They belong to the "novel genomic techniques (NGT)", defined as “techniques capable to change the genetic material of an organism and that have emerged or have been developed since 2001”. The possible detection of genome-edited GMOs depends on the type of modification achieved and whether (transgenic) foreign DNA or genome-editing compo-nents may have been integrated into the plant genome. For detection, small mutations (point mutations) pose the greatest challenge. In this report, based on the currently used methods for the detection of classical GMOs, the existing possibilities and challenges for genome-edited plants are discussed.
Detection of even very small sequence differences, such as point mutations, is possible with the current technical equipment of a control laboratory. Optimization steps (primer/probe de-sign, thermal profiles) may be required to increase sensitivity. To transfer detection methods into routine operation, the methods have to be validated. A characteristic-unique modification or the combined detection of multiple modifications in a tested genome may allow the identi-fication of genome-edited plants. Quantification of these analytical results by currently avail-able methods is technically possible using certain approaches.
Accurate information on the modification made is the most important prerequisite for the de-velopment of methods for detection, identification and quantification of genome-edited GMOs. Such information could be gathered from publicly available sources. It is recom-mended combining information from different public documents, including scientific publica-tions, patents and regulatory documents from third countries. This information could be gath-ered in databases maintained by international organizations. Existing databases, such as the Biosafety Clearing House under the Cartagena Protocol on Biosafety or EUginius, could serve as a model for an international database for sharing information on globally marketed NGT products. It is recommended to establish such a database, maintained at the interna-tional level, to provide easy access for control laboratories to all relevant information.
In order to be able to establish the developed methods in the laboratory, and for control pur-poses, reference material is necessary (biological material and derived material; includes the genome-edited plant and/or the parental line/starting material). Upon application for food, feed, or cultivation purposes, the applicant shall provide reference material. For GMO without application, the availability of reference material can be ensured by a central body (e.g., the Joint Research Centre, JRC) in cooperation with the developers. If biological material is not available, plasmids with the corresponding DNA sequences can also be synthesized based on sequence information, if available, and serve as reference material.
Research is needed primarily in method development, characterization of genome editing applications (non-intended modifications, specificities, recognition sequences, etc.), and in the development of databases for pan-genomes to be used in control laboratories in the long term. It is recommended that this work be funded through European research funding pro-grammes and through mandates to JRC and EFSA.
... The use of different targets and signal amplification of variants, with or without hybridisation, makes available a range of qualitative and quantitative methods (QRT-PCR, LAMP, NASBA, LCR, SNPLex…) that can be chosen, depending on the target. These methodological approaches can be applied from the field to the laboratory, from point mutation to the junction sequence between rearranged elements and from single to multiple targets Bertheau, 2019;Chhalliyil et al., 2020). Associated with various decision support tools, they allow, via the matrix approach, the identification of any GMO. ...
... For others, the appearance of off-target effects such as tumors in humans (Haapaniemi et al., 2018) calls for caution in the use of these tools. Moreover, studies have traced changes caused by these technologies and thus attributed property rights to final products (Bertheau, 2019). ...
The evolution of varietal creation methods led in 2012 to the advent of the genome editing technique, CRISPR-CaS9. This technique would make it possible to create new varieties quickly and cheaply. Although some consider CRISPR-CaS9 to be revolutionary, others consider it a potential societal threat. To document the controversy, we explain the socioeconomic conditions under which this technique could be accepted for the creation of a rainfed rice variety in Madagascar. The methodological framework is based on 38 individual and semistructured interviews, a multistakeholder forum with organizations interviewed, and a survey of 148 rice producers. Results reveal that the acceptability of genome editing requires (i) strengthening the seed system through the operationalization of regulatory structures and the upgrading of stakeholders' knowledge of genetically modified organisms, (ii) assessing the effects of the edited variety on biodiversity and soil nitrogen dynamics, and (iii) strengthening the technical and human capacities of the biosafety body. Structural mechanisms for regulating the seed system are necessary to ensure safe experimentation of genome editing techniques. Organizational innovation also appears to be necessary. The study documents how collective learning between communities of scientists and nonscientists is a component of systemic processes of varietal innovation.
... If adequate sequence information is made available, distinct, unique genomic changes can be analytically detected [22]. These changes may either be (several) intended mutations or a combination of intended mutations with unintended changes that are simultaneously present in a genome-edited plant [23][24][25]. Plant genes with a low genetic distance may not be separable, a phenomenon frequently observed in classical breeding [26]. Similarly, mutations are expected to be genetically linked if located in the immediate vicinity. ...
It is difficult to trace and identify genome-edited food and feed products if relevant information is not made available to competent authorities. This results in major challenges, as genetically modified organism (GMO) regulatory frameworks for food and feed that apply to countries such as the member states of the European Union (EU) require enforcement based on detection. An international anticipatory detection and identification framework for voluntary collaboration and collation of disclosed information on genome-edited plants could be a valuable tool to address these challenges caused by data gaps. Scrutinizing different information sources and establishing a level of information that is sufficient to unambiguously conclude on the application of genome editing in the plant breeding process can support the identification of genome-edited products by complementing the results of analytical detection. International coordination to set up an appropriate state-of-the-art database is recommended to overcome the difficulty caused by the non-harmonized bio-safety regulation requirements of genome-edited food and feed products in various countries. This approach helps to avoid trade disruptions and to facilitate GMO/non-GMO labeling schemes. Implementation of the legal requirements for genome-edited food and feed products in the EU and elsewhere would substantially benefit from such an anticipatory framework.
... Whilst protoplast and tissue culture based processes are already prone to induce mutations (see also line 326), there is additionally the possibility of the uptake and integration of any DNA sequences present in the medium, were they to be present from the ribonucleoprotein production (insufficient purification). See also Bertheau (2019). ...
The European Food Safety Authority (EFSA) carried out a public consultation to receive input from interested parties on the applicability of the EFSA Opinion on site‐directed nucleases type 3 for the safety assessment of plants developed using site‐directed nucleases type 1 and 2 and oligonucleotide‐directed mutagenesis. This draft scientific opinion was prepared by the GMO Panel, supported by the Working Group on Molecular Characterization. The draft opinion was endorsed by the EFSA GMO Panel for public consultation on 1stApril 2020. The written public consultation was open from 15April 2020 until 5 June 2020. EFSA received comments from 51 different interested parties. EFSA and its GMO Panel wish to thank all stakeholders for their contributions to this work. The present report contains the comments received and details how they have been considered for finalisation of the opinion. The final opinion was adopted at the GMO Panel Plenary meeting on 14October 2020 and will be published in the EFSA Journal.© European Food Safety Authority, 2020
... A key area of controversy is whether the product or the process is relevant for assessment and approval, because the products of NBTs are sometimes not distinguishable from those of conventional breeding methods [11]. The issue of detection remains controversial, and there have been calls for further research on this topic before NBTs are approved for commercial application [12]. Furthermore, while there was a legal obligation for developers and users of MON810 to limit 'contamination', i.e., the spread of transgenes, some products of NBTs incorporate 'gene drives', which are specifically designed to enable a particular genome to be propagated throughout a population [13]. ...
The paper uses qualitative interviews and document analysis to examine conflicts over plant and animal breeding techniques from the perspectives of Social and Political Ecology. It asks how past conflicts over genetically modified organisms (GMOs) can inform understandings of possible trajectories of emerging conflicts over new breeding techniques (NBTs) such as CRISPR/Cas genome editing. Case studies of conflicts in three areas where the transgenic maize MON810 was cultivated in Germany from 2005–2008 show that the escalation of conflict coincided with the first tangible presence of these already controversial organisms in the rural landscape. Location-specific interlinkages between discursive and material dimensions gave rise to different pathways of conflict in the three areas studied. These empirical results inform the analysis of emerging conflicts over NBTs in Germany and the United Kingdom. The future of NBTs in both countries is still open, and the divergence of regulatory frameworks in Europe could lead to the development of ‘NBT hotspots’ located in particular European countries, provoking an escalation of conflict in areas where commercial application takes place. The paper concludes by examining the potential for a politicization of future conflicts to encompass wider issues related to the transformation of agricultural systems towards sustainability.
Despite rapid advances in sequencing technologies, accurately calling genetic variants present in an individual genome from billions of short, errorful sequence reads remains challenging. Here we show that a deep convolutional neural network can call genetic variation in aligned next-generation sequencing read data by learning statistical relationships between images of read pileups around putative variant and true genotype calls. The approach, called DeepVariant, outperforms existing state-of-the-art tools. The learned model generalizes across genome builds and mammalian species, allowing nonhuman sequencing projects to benefit from the wealth of human ground-truth data. We further show that DeepVariant can learn to call variants in a variety of sequencing technologies and experimental designs, including deep whole genomes from 10X Genomics and Ion Ampliseq exomes, highlighting the benefits of using more automated and generalizable techniques for variant calling.
The diversity of somatic mutations in human cancers can be decomposed into individual mutational signatures, patterns of mutagenesis that arise because of DNA damage and DNA repair processes that have occurred in cells as they evolved towards malignancy. Correlations between mutational signatures and environmental exposures, enzymatic activities and genetic defects have been described, but human cancers are not ideal experimental systems-the exposures to different mutational processes in a patient's lifetime are uncontrolled and any relationships observed can only be described as an association. Here, we demonstrate the proof-of-principle that it is possible to recreate cancer mutational signatures in vitro using CRISPR-Cas9-based gene-editing experiments in an isogenic human-cell system. We provide experimental and algorithmic methods to discover mutational signatures generated under highly experimentally-controlled conditions. Our in vitro findings strikingly recapitulate in vivo observations of cancer data, fundamentally validating the concept of (particularly) endogenously-arising mutational signatures.
CRISPR-Cas9 transcriptional repressors have emerged as robust tools for disrupting gene regulation in vitro but have not yet been adapted for systemic delivery in adult animal models. Here we describe a Staphylococcus aureus Cas9-based repressor (dSaCas9KRAB) compatible with adeno-associated viral (AAV) delivery. To evaluate dSaCas9KRAB efficacy for gene silencing in vivo, we silenced transcription of Pcsk9, a regulator of cholesterol levels, in the liver of adult mice. Systemic administration of a dual-vector AAV8 system expressing dSaCas9KRAB and a Pcsk9-targeting guide RNA (gRNA) results in significant reductions of serum Pcsk9 and cholesterol levels. Despite a moderate host response to dSaCas9KRAB expression, Pcsk9 repression is maintained for 24 weeks after a single treatment, demonstrating the potential for long-term gene silencing in post-mitotic tissues with dSaCas9KRAB. In vivo programmable gene silencing enables studies that link gene regulation to complex phenotypes and expands the CRISPR-Cas9 perturbation toolbox for basic research and gene therapy applications.
Here we analyse genetic variation, population structure and diversity among 3,010 diverse Asian cultivated rice (Oryza sativa L.) genomes from the 3,000 Rice Genomes Project. Our results are consistent with the five major groups previously recognized, but also suggest several unreported subpopulations that correlate with geographic location. We identified 29 million single nucleotide polymorphisms, 2.4 million small indels and over 90,000 structural variations that contribute to within- and between-population variation. Using pan-genome analyses, we identified more than 10,000 novel full-length protein-coding genes and a high number of presence-absence variations. The complex patterns of introgression observed in domestication genes are consistent with multiple independent rice domestication events. The public availability of data from the 3,000 Rice Genomes Project provides a resource for rice genomics research and breeding.
Structural variants (SVs) can result in changes in gene expression due to abnormal chromatin folding and cause disease. However, the prediction of such effects remains a challenge. Here we present a polymer-physics-based approach (PRISMR) to model 3D chromatin folding and to predict enhancer-promoter contacts. PRISMR predicts higher-order chromatin structure from genome-wide chromosome conformation capture (Hi-C) data. Using the EPHA4 locus as a model, the effects of pathogenic SVs are predicted in silico and compared to Hi-C data generated from mouse limb buds and patient-derived fibroblasts. PRISMR deconvolves the folding complexity of the EPHA4 locus and identifies SV-induced ectopic contacts and alterations of 3D genome organization in homozygous or heterozygous states. We show that SVs can reconfigure topologically associating domains, thereby producing extensive rewiring of regulatory interactions and causing disease by gene misexpression. PRISMR can be used to predict interactions in silico, thereby providing a tool for analyzing the disease-causing potential of SVs.
Genetic toxicology assays estimate mutation frequencies by phenotypically screening for the activation or inactivation of endogenous or exogenous reporter genes. These reporters can only detect mutations in narrow areas of the genome and their use is often restricted to certain in vitro and in vivo models. Here, we show that Interclonal Genetic Variation (ICGV) can directly identify mutations genome-wide by comparing sequencing data of single-cell clones derived from the same source or organism. Upon ethyl methanesulfonate (EMS) exposure, ICGV detected greater levels of mutation in a dose- and time-dependent manner in E. coli. In addition, ICGV was also able to identify a ∼20-fold increase in somatic mutations in T-cell clones derived from an N-ethyl-N-nitrosourea (ENU)-treated rat vs. a vehicle-treated rat. These results demonstrate that the genetic differences of single-cell clones can be used for genome-wide mutation detection.
Background
Next-Generation Sequencing (NGS) has transformed the life sciences and many research groups are newly dependent upon computer clusters to store and analyse large datasets. This creates challenges for e-infrastructures accustomed to hosting computationally mature research in other sciences. Using data gathered from our own clusters at UPPMAX computing centre at Uppsala University, Sweden, where core hours usage by ∼800 NGS and ∼200 non-NGS projects is now similar, we compare and contrast the growth, administrative burden and cluster usage of NGS projects with projects from other sciences.
Results
The number of NGS projects has grown rapidly since 2010, with growth driven by entry of new research groups. Storage used by NGS projects has grown more rapidly since 2013 and is now limited by disk capacity. NGS users submit nearly twice as many support tickets per user, and 11 more tools are installed each month for NGS than non-NGS projects. We develop usage and efficiency metrics and show that compute jobs in NGS projects use more RAM than in non-NGS projects, are more variable in core usage, and rarely span multiple nodes. NGS jobs use booked resources less efficiently for a variety of reasons. Active monitoring can improve this somewhat.
Conclusions
Hosting NGS projects imposes a large administrative burden at UPPMAX, due to large numbers of inexperienced users and diverse and rapidly evolving research areas. We give a set of recommendations for e-infrastructures hosting NGS research projects. We provide anonymised versions of our storage, job and efficiency databases.
In the last decade, RNA interference (RNAi), a cellular mechanism that uses RNA-guided degradation of messenger RNA transcripts, has had an important impact on identifying and characterizing gene function. First discovered inCaenorhabditis elegans, RNAi can be used to silence the expression of genes through introduction of exogenous double-stranded RNA into cells. InDrosophila, RNAi has been applied in cultured cells orin vivoto perturb the function of single genes or to systematically probe gene function on a genome-wide scale. In this review, we will describe the use of RNAi to study gene function inDrosophilawith a particular focus on high-throughput screening methods applied in cultured cells. We will discuss available reagent libraries and cell lines, methodological approaches for cell-based assays, and computational methods for the analysis of high-throughput screens. Furthermore, we will review the generation and use of genome-scale RNAi libraries for tissue-specific knockdown analysisin vivoand discuss the differences and similarities with the use of genome-engineering methods such as CRISPR/Cas9 for functional analysis.
The selection of the best-fit-for-purpose analytical method to be implemented in the laboratory is difficult due to availability of multiple methods, targets, aims of detection, and different kinds and sources of more or less reliable information. Several factors, such as method performance, practicability, cost of setup, and running costs need to be considered together with personnel training when selecting the most appropriate method. The aim of our work was to prepare a flexible multicriteria decision analysis model suitable for evaluation and comparison of analytical methods used for the purpose of detecting and/or quantifying genetically modified organisms, and to use this model to evaluate a variety of changing analytical methods. Our study included selection of PCR-, isothermal-, protein-, microarray-, and next-generation sequencing-based methods in simplex and/or multiplex formats. We show that the overall result of their fitness for purpose is relatively similar; however, individual criteria or a group of related criteria exposed more substantial differences between the methods. The proposed model of this decision support system enables easy modifications and is thus suitable for any other application of complex analytical methods.
Methylated bases in tRNA, rRNA and mRNA control a variety of cellular processes, including protein synthesis, antimicrobial resistance and gene expression. Currently, bulk methods that report the average methylation state of ~10 4-10 7 cells are used to detect these modifications, obscuring potentially important biological information. Here, we use in situ hybridization of Molecular Beacons for single-cell detection of three methylations (m 6 2 A, m 1 G and m 3 U) that destabilize Watson-Crick base pairs. Our method-methylation-sensitive RNA fluorescence in situ hybridization-detects single methylations of rRNA, quantifies antibiotic-resistant bacteria in mixtures of cells and simultaneously detects multiple methylations using multi-color fluorescence imaging.
Detection of somatic mutations holds great potential in cancer treatment and has been a very active research field in the past few years, especially since the breakthrough of the next-generation sequencing technology. A collection of variant calling pipelines have been developed with different underlying models, filters, input data requirements, and targeted applications. This review aims to enumerate these unique features of the state-of-the-art variant callers, in the hope to provide a practical guide for selecting the appropriate pipeline for specific applications. We will focus on the detection of somatic single nucleotide variants, ranging from traditional variant callers based on whole genome or exome sequencing of paired tumor-normal samples to recent low-frequency variant callers designed for targeted sequencing protocols with unique molecular identifiers. The variant callers have been extensively benchmarked with inconsistent performances across these studies. We will review the reference materials, datasets, and performance metrics that have been used in the benchmarking studies. In the end, we will discuss emerging trends and future directions of the variant calling algorithms.
As a part of the ELIXIR-EXCELERATE efforts in capacity building, we present here 10 steps to facilitate researchers getting started in genome assembly and genome annotation. The guidelines given are broadly applicable, intended to be stable over time, and cover all aspects from start to finish of a general assembly and annotation project.
Intrinsic properties of genomes are discussed, as is the importance of using high quality DNA. Different sequencing technologies and generally applicable workflows for genome assembly are also detailed. We cover structural and functional annotation and encourage readers to also annotate transposable elements, something that is often omitted from annotation workflows. The importance of data management is stressed, and we give advice on where to submit data and how to make your results Findable, Accessible, Interoperable, and Reusable (FAIR).
Objectives
We aimed at identifying genes related to hereditary type 2 diabetes expressed in the liver and the adipose tissue of spontaneous diabetic gerbils using suppression subtractive hybridization (SSH) screening.
Methods
Two gerbil littermates, one with high and the other with normal blood glucose level, from our previously bred spontaneous diabetic gerbil strain were used in this study. To identify differentially expressed genes in the liver and the adipose tissue, mRNA from these tissues was extracted and SSH libraries were constructed for screening. After sequencing and BLAST analyzing, up or down-regulated genes possibly involved in metabolism and diabetes were selected, and their expression levels in diabetic gerbils and normal controls were analyzed using quantitative RT-PCR and Western blotting.
Results
A total of 4 SSH libraries were prepared from the liver and the adipose tissue of gerbils. There are 95 up or down-regulated genes were identified to be involved in metabolism, oxidoreduction, RNA binding, cell proliferation, and differentiation or other function. Expression of 17 genes most possibly associated with diabetes was analyzed and seven genes (Sardh, Slc39a7, Pfn1, Arg1, Cth, Sod1 and P4hb) in the liver and one gene (Fabp4) in the adipose tissue were identified that were significantly differentially expressed between diabetic gerbils and control animals.
Conclusions
We identified eight genes associated with type 2 diabetes from the liver and the adipose tissue of gerbils via SSH screening. These findings provide further insights into the molecular mechanisms of diabetes and imply the value of our spontaneous diabetic gerbil strain as a diabetes model.
Dodders (Cuscuta spp.) are obligate parasitic plants that obtain water and nutrients from the stems of host plants via specialized feeding structures called haustoria. Dodder haustoria facilitate bidirectional movement of viruses, proteins and mRNAs between host and parasite, but the functional effects of these movements are not known. Here we show that Cuscuta campestris haustoria accumulate high levels of many novel microRNAs (miRNAs) while parasitizing Arabidopsis thaliana. Many of these miRNAs are 22 nucleotides in length. Plant miRNAs of this length are uncommon, and are associated with amplification of target silencing through secondary short interfering RNA (siRNA) production. Several A. thaliana mRNAs are targeted by 22-nucleotide C. campestris miRNAs during parasitism, resulting in mRNA cleavage, secondary siRNA production, and decreased mRNA accumulation. Hosts with mutations in two of the loci that encode target mRNAs supported significantly higher growth of C. campestris. The same miRNAs that are expressed and active when C. campestris parasitizes A. thaliana are also expressed and active when it infects Nicotiana benthamiana. Homologues of target mRNAs from many other plant species also contain the predicted target sites for the induced C. campestris miRNAs. These data show that C. campestris miRNAs act as trans-species regulators of host-gene expression, and suggest that they may act as virulence factors during parasitism.
Seed, a unit of plant reproduction, is an important source of food, feed, and feedstock. Seed size is a key component of a seed yield trait. Improving seed size has been one of the primary goals of plant breeding since the domestication of crop plants. In the last one and half decade, molecular studies on seed development have gained a great importance in plant research and as a result, a number of molecular regulators of seed size have been identified in the model plant Arabidopsis and some of the crop plants. Seed development is a complex process involving a coordinated growth of maternal and zygotic tissues, which is regulated by the integrated action of transcriptional, epigenetic, hormonal, peptide and sugar signaling regulators. In this review, the role of different transcriptional, epigenetic, hormonal, peptide and sugar signaling regulators in seed development pathways are discussed in detail followed by a brief description of the application of the knowledge gained over the years on molecular regulation of seed development for engineering seed size in crop plants using novel transgenic and recently evolved new breeding strategies such as genome editing.
Genome editing is one of the most promising biotechnologies to improve crop performance. Common wheat is a staple food for mankind. In the past few decades both basic and applied research on common wheat has lagged behind other crop species due to its complex, polyploid genome and difficulties in genetic transformation. Recent breakthroughs in wheat transformation permit a revolution in wheat biotechnology. In this review, we summarize recent progress in wheat genetic transformation and its potential for wheat improvement. We then review recent progress in plant genome editing, which is now readily available in wheat. We also discuss measures to further increase transformation efficiency and potential applications of genome editing in wheat. We propose that, together with a high quality reference genome, the time for efficient genetic engineering and functionality studies in common wheat has arrived.
Because plants do not possess a defined germline, deleterious somatic mutations can be passed to gametes, and a large number of cell divisions separating zygote from gamete formation may lead to many mutations in long-lived plants. We sequenced the genome of two terminal branches of a 234-year-old oak tree and found several fixed somatic single-nucleotide variants whose sequential appearance in the tree could be traced along nested sectors of younger branches. Our data suggest that stem cells of shoot meristems in trees are robustly protected from the accumulation of mutations.
ELife digest
Most plants spend their entire lives in one fixed spot and so must be able to quickly adapt to any changes in their surroundings. For example, high levels of salt in the soil – which can be toxic to cells – triggers stress responses in plants that help them to mitigate any damage. Once the stress has passed, plants are able to retain a memory of it, which allows them to respond more quickly if they face the same stress in future. Furthermore, plants may pass on this ‘stress memory’ to their offspring.
It is thought that stress memory is programmed by chemical modifications to DNA known as epigenetic marks. These marks do not alter the genetic information that is encoded by the DNA itself, but they can change the activity of particular genes. Environmental stress leads to changes in the epigenetic marks found on many plant genes, which can be directly passed on from the parent plant to its offspring. However, it was not clear whether the epigenetic marks that programme stress memory can be passed on in this way.
Wibowo, Becker et al. investigated how a model plant called Arabidopsis thaliana is able to remember periods of salt stress. The experiments show that high levels of salt can trigger changes in the patterns of epigenetic marks associated with particular regions of DNA. This memory is reinforced by repetitive exposure to similar salt stress and can be passed onto offspring, primarily through the maternal line. However, this stress memory is not fixed in future generations as the epigenetic marks can be reset to their original patterns if plants find themselves growing and reproducing under non-stress conditions.
In sum, the findings of Wibowo, Becker et al. show that epigenetic marks allow plants to inherit stress memory on a temporary basis while the stress is present, but to gradually lose the memory if the stress does not return. Future studies will focus on finding out if stress memory in crop plants works in the same way.
Main conclusion: The transcripts of transgenic prosystemin (PS) gene are mobile and the PS mRNA can be translated into protein in tomato and tobacco plants. Systemin (SYS) and its precursor protein, prosystemin (PS), are upstream components of the wound-induced signaling pathway in tomato. Although the mobile signal(s) for wound responses has been the subject of considerable research, its identity remains controversial. Intensive studies have revealed the essential role of mRNA on plant systemic signaling. We hypothesize that PS mRNA can act as a transmissible signal in tomato. Herein, we demonstrated that transgenic PS mRNA occurs in leaves located at considerable distances from the initial site of its generation by a transient Agrobacterium-infiltration assay system. We also showed that PS protein is present in the vascular bundle of the distant leaves. Our results indicate that transgenic PS mRNA may be functional as a long-distance signal to modulate systemic defense responses in tomato, providing novel insights into the multifaceted systems by which SYS signaling transports.
Genomic alterations driving tumorigenesis result from the interaction of environmental exposures and endogenous cellular processes. With a diversity of risk factors, liver cancer is an ideal model to study these interactions. Here, we analyze the whole genomes of 44 new and 264 published liver cancers and we identify 10 mutational and 6 structural rearrangement signatures showing distinct relationships with environmental exposures, replication, transcription, and driver genes. The liver cancer-specific signature 16, associated with alcohol, displays a unique feature of transcription-coupled damage and is the main source of CTNNB1 mutations. Flood of insertions/deletions (indels) are identified in very highly expressed hepato-specific genes, likely resulting from replication-transcription collisions. Reconstruction of sub-clonal architecture reveals mutational signature evolution during tumor development exemplified by the vanishing of aflatoxin B1 signature in African migrants. Finally, chromosome duplications occur late and may represent rate-limiting events in tumorigenesis. These findings shed new light on the natural history of liver cancers.
Rice is one of the most important staple crops, second largest produced
cereal, feeding more than half of the world’s population. Rice is subjected
to important diseases which dramatically reduce crop yields up to 30%.
Biotic factors that damages paddy crop are virus, bacteria, fungi, nematode,
and insect pests. There are several methods of rice disease management
but all existing methods have some limitations. RNA interference
(RNAi)-induced gene silencing emerged as an effective tool to engineer
resistant plants to different types of biotic stresses. This chapter emphasizes
on different types of biotic factors that negatively affects the rice production.
The available methods of biotic stress management are discussed
along with their limitations. RNAi-mediated management of rice diseases
has some advantages over existing disease management programs. This
approach can also be used as a potential tool for disease management
of other commercially important crops which are highly susceptible to
various types of biotic stresses.
Assisted reproductive therapies (ART) have become increasingly common worldwide and numerous retrospective studies have indicated that ART-conceived children are more likely to develop the overgrowth syndrome Beckwith-Wiedemann (BWS). In bovine, the use of ART can induce a similar overgrowth condition, which is referred to as large offspring syndrome (LOS). Both BWS and LOS involve misregulation of imprinted genes. However, it remains unknown whether molecular alterations at non-imprinted loci contribute to these syndromes. Here we examined the transcriptome of skeletal muscle, liver, kidney, and brain of control and LOS bovine fetuses and found that different tissues within LOS fetuses have perturbations of distinct gene pathways. Notably, in skeletal muscle, multiple pathways involved in myoblast proliferation and fusion into myotubes are misregulated in LOS fetuses. Further, characterization of the DNA methylome of skeletal muscle demonstrates numerous local methylation differences between LOS and controls; however, only a small percent of differentially expressed genes (DEGs), including the imprinted gene IGF2R, could be associated with the neighboring differentially methylated regions. In summary, we not only show that misregulation of non-imprinted genes and loss-of-imprinting characterize the ART-induced overgrowth syndrome but also demonstrate that most of the DEGs is not directly associated with DNA methylome epimutations.
Quantitative or complex traits are controlled by many genes and environmental factors. Most traits in livestock breeding are quantitative traits. Mapping genes and causative mutations generating the genetic variance of these traits is still a very active area of research in livestock genetics. Since genome-wide and dense SNP panels are available for most livestock species, genome-wide association studies (GWASs) have become the method of choice in mapping experiments. Different statistical models are used for GWASs. We will review the frequently used single-marker models and additionally describe Bayesian multi-marker models. The importance of nonadditive genetic and genotype-by-environment effects along with GWAS methods to detect them will be briefly discussed. Different mapping populations are used and will also be reviewed. Whenever possible, our own real-data examples are included to illustrate the reviewed methods and designs. Future research directions including post-GWAS strategies are outlined.
Gulf War Illness (GWI) is a chronic multi-symptom illness not currently diagnosed by standard medical or laboratory test that affects 30% of veterans who served during the 1990–1991 Gulf War. The clinical presentation of GWI is comparable to that of patients with certain mitochondrial disorders–i.e., clinically heterogeneous multisystem symptoms. Therefore, we hypothesized that mitochondrial dysfunction may contribute to both the symptoms of GWI as well as its persistence over time. We recruited 21 cases of GWI (CDC and Kansas criteria) and 7 controls to participate in this study. Peripheral blood samples were obtained in all participants and a quantitative polymerase chain reaction (QPCR) based assay was performed to quantify mitochondrial and nuclear DNA lesion frequency and mitochondrial DNA (mtDNA) copy number (mtDNAcn) from peripheral blood mononuclear cells. Samples were also used to analyze nuclear DNA lesion frequency and enzyme activity for mitochondrial complexes I and IV. Both mtDNA lesion frequency (p = 0.015, d = 1.13) and mtDNAcn (p = 0.001; d = 1.69) were elevated in veterans with GWI relative to controls. Nuclear DNA lesion frequency was also elevated in veterans with GWI (p = 0.344; d = 1.41), but did not reach statistical significance. Complex I and IV activity (p > 0.05) were similar between groups and greater mtDNA lesion frequency was associated with reduced complex I (r² = -0.35, p = 0.007) and IV (r² = -0.28, p < 0.01) enzyme activity. In conclusion, veterans with GWI exhibit greater mtDNA damage which is consistent with mitochondrial dysfunction.
Agrobacterium rhizogenes causes hairy root growth on a large number of plant species. It does so by transferring specific DNA fragments (T-DNA) from its root-inducing plasmid (pRi) into plant cells. Expression of T-DNA genes leads to abnormal root growth and production of specific metabolites (opines) which are taken up by the bacterium and used for its growth. Recent work has shown that several Nicotiana, Linaria, and Ipomoea species contain T-DNA genes from A. rhizogenes in their genomes. Plants carrying such T-DNAs (called cellular T-DNA or cT-DNA) can be considered as natural transformants. In the Nicotiana genus, seven different T-DNAs are found originating from different Agrobacterium strains, and in the Tomentosae section no <4 successive insertion events took place. In several cases cT-DNA genes were found to be expressed. In some Nicotiana tabacum cultivars the opine synthesis gene TB-mas2′ is expressed in the roots. These cultivars were found to produce opines. Here we review what is known about natural Agrobacterium transformants, develop a theoretical framework to analyze this unusual phenomenon, and provide some outlines for further research.
The oil overlay micro-drop system is widely used for cultures of mammalian gametes and embryos. We evaluated hereby the effects of two unaltered commercial oils— Sigma mineral oil (S-MO) and Nidoil paraffin oil (N-PO)—on in vitro embryo production (IVP) outcomes using a pig model. The results showed that while either oil apparently did not affect oocyte maturation and fertilization rates, S-MO negatively affected embryo cleavage rates, blastocyst formation rates, and, consequently, total blastocyst efficiency of the system. No differences in the oxidation state were found between the oils or culture media incubated under S-MO or N-PO. Although both oils slightly differed in elemental composition, there were no differences in the concentrations of elements between fresh media and media incubated under oils. By contrast, we demonstrated clear oil-type differences in both the composition of volatile organic compounds (VOC) and the transfer of some of these VOC´s (straight-chain alkanes and pentanal and 1,3-diethyl benzene) to the culture medium, which could have influenced embryonic development.
Today, there is a collection of a tremendous amount of bio-data because of the computerized applications worldwide. Therefore, scholars have been encouraged to develop effective methods to extract the hidden knowledge in these data. Consequently, a challenging and valuable area for research in artificial intelligence has been created. Bioinformatics creates heuristic approaches and complex algorithms using artificial intelligence and information technology in order to solve biological problems. Intelligent implication of the data can accelerate biological knowledge discovery. Data mining, as biology intelligence, attempts to find reliable, new, useful and meaningful patterns in huge amounts of data. Hence, there is a high potential to raise the interaction between artificial intelligence and bio-data mining. The present paper argues how artificial intelligence can assist bio-data analysis and gives an up-to-date review of different applications of bio-data mining. It also highlights some future perspectives of data mining in bioinformatics that can inspire further developments of data mining instruments. Important and new techniques are critically discussed for intelligent knowledge discovery of different types of row datasets with applicable examples in human, plant and animal sciences. Finally, a broad perception of this hot topic in data science is given.
Among several smallRNAs classes, microRNAs play an important role in controlling the post-transcriptional events. Next generation sequencing has played a major role in extending the landscape of miRNAs and revealing their spatio-temporal roles in development and abiotic stress. Lateral evolution of these smallRNAs classes have widely been seen with the recently emerging knowledge on tRNA derived smallRNAs. In the present perspective, we discussed classification, identification and roles of tRNA derived smallRNAs across plants and their potential involvement in abiotic and biotic stresses.
Biological material is at the forefront of research programs, as well as application fields such as breeding, aquaculture, and reforestation. While sophisticated techniques are used to produce this material, all too often, there is no strict monitoring during the “production” process to ensure that the specific varieties are the expected ones. Confidence rather than evidence is often applied when the time comes to start a new experiment or to deploy selected varieties in the field. During the last decade, genomics research has led to the development of important resources, which have created opportunities for easily developing tools to assess the conformity of the material along the production chains. In this study, we present a simple methodology that enables the development of a traceability system which, is in fact a by-product of previous genomic projects. The plant production system in white spruce (Picea glauca) is used to illustrate our purpose. In Quebec, one of the favored strategies to produce elite varieties is to use somatic embryogenesis (SE). In order to detect human errors both upstream and downstream of the white spruce production process, this project had two main objectives: (i) to develop methods that make it possible to trace the origin of plants produced, and (ii) to generate a unique genetic fingerprint that could be used to differentiate each embryogenic cell line and ensure its genetic monitoring. Such a system had to rely on a minimum number of low-cost DNA markers and be easy to use by non-specialists. An efficient marker selection process was operationalized by testing different classification methods on simulated datasets. These datasets were generated using in-house bioinformatics tools that simulated crosses involved in the breeding program for which genotypes from hundreds of SNP markers were already available. The rate of misidentification was estimated and various sources of mishandling or contamination were identified. The method can easily be applied to other production systems for which genomic resources are already available.
Scientists have discovered various prognostic gene signatures (GSs) in different cancer types. Surprisingly, although different GSs from the same cancer type can be used to measure similar biological characteristics, often rarely is there a gene shared by different GSs. To explain such a paradox, we hypothesized that GSs from the same cancer type may be regulated by common regulatory motifs. To test this hypothesis, we carried out a comprehensive motif analysis on the prognostic GSs from five cancer types. We demonstrated that GSs from individual cancer type as well as across cancer types share regulatory motifs. We also observed that transcription factors that likely bind to these shared motifs have prognostic functions in cancers. Moreover, 75% of the predicted cofactors of these transcription factors may have cancer-related functions and some cofactors even have prognostic functions. In addition, there exist common microRNAs that regulate different GSs from individual cancer types and across cancer types, several of which are prognostic biomarkers for the corresponding cancer types. Our study suggested the existence of common regulatory mechanisms shared by GSs from individual cancer types and across cancer types, which shed light on the discovery of new prognostic GSs in cancers and the understanding of the regulatory mechanisms of cancers.
Genome editing by the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR-associated protein 9) system is a revolutionary strategy to study gene functions. Since the efficiency of gene disruption in cell culture does not reach 100% typically, cloning of mutant cells is often performed to obtain fully mutated cells. Therefore, a method to discriminate accurately mutated clones easily and quickly is crucial to accelerate the research using CRISPR/Cas9. Here, we show that knockout cells can be discriminated by a competition-based PCR, using a mixture of three primers, among which one primer overlaps with the Cas9 cleavage site. Together, we show how to optimize primer design in order to improve the effectiveness of the discrimination. Finally, we applied this method to show that mutations conferring drug resistance can be detected with high accuracy. The provided method is easy to perform and requires only basic laboratory equipment, making it suitable for almost all laboratories.
The 4D Nucleome Network aims to develop and apply approaches to map the structure and dynamics of the human and mouse genomes in space and time with the goal of gaining deeper mechanistic insights into how the nucleus is organized and functions. The project will develop and benchmark experimental and computational approaches for measuring genome conformation and nuclear organization, and investigate how these contribute to gene regulation and other genome functions. Validated experimental technologies will be combined with biophysical approaches to generate quantitative models of spatial genome organization in different biological states, both in cell populations and in single cells.
A woman who is healthy at the time of conception is more likely to have a successful pregnancy and a healthy child. We reviewed published evidence and present new data from high, low and middle income countries on the timing and importance of preconception health for subsequent maternal and child health. We describe the extent to which pregnancy is planned, and whether planning is linked to preconception health behaviours.
Observational studies show strong links between health before pregnancy and maternal and child health outcomes, with consequences that can extend across generations, but awareness of these links is not widespread. Poor nutrition and obesity are rife among women of reproductive age, and differences between high and lower income countries have become less distinct, with typical diets falling far short of nutritional recommendations in both settings and especially among adolescents.
Numerous studies show that micronutrient supplementation starting in pregnancy can correct important maternal nutrient deficiencies, but effects on child health outcomes are disappointing. Other interventions to improve diet during pregnancy have had little impact on maternal and newborn health outcomes. There have been comparatively few attempts at preconception diet and lifestyle intervention. Improvements in the measurement of pregnancy planning have quantified the degree of pregnancy planning and suggest that this is more common than previously recognised. Planning for pregnancy is associated with a mixed pattern of health behaviours before conception.
We propose novel definitions of the preconception period relating to embryo development and to action at individual or population level. A sharper focus on intervention before conception is needed to improve maternal and child health and reduce the growing burden of non-communicable disease. Alongside continued efforts to reduce smoking, alcohol and obesity in the population, we call for heightened awareness of preconception health, particularly regarding diet and nutrition. Importantly health professionals should be alerted to ways of identifying women who are planning a pregnancy. <br/
Gene-editing tool can be harnessed to give a close-up view of life's most basic processes.
Maps of defense arsenals in microbial genomes
To survive the attack of foreign invaders such as viruses and plasmids, bacteria and archaea fight back with immune systems that are usually clustered in “defense islands” in their genomes. Doron et al. took advantage of this property to map microbial defense systems systematically (see the Perspective by Kim). Candidate immune systems were then experimentally validated for their activities. Like well-known defense arsenals such as restriction-modification and CRISPR systems, these additional immune systems now require mechanistic investigation and could potentially be engineered into useful molecular tools in the future.
Science , this issue p. eaar4120 ; see also p. 993
A CRISPR device to record time
The CRISPR adaptation system has been used to record the sequence and ordering of exogenous oligonucleotides that are electroporated into cell populations. Sheth et al. engineered a system bypassing the use of exogenous DNA to directly record temporal signals. An input biological signal is transformed into the ratio of the frequency of incorporating trigger DNA to that of incorporating reference DNA into the genomes of a bacterial population. A multiplexing strategy enables simultaneous recording of three environmental signals with high temporal resolution.
Science , this issue p. 1457
Genome editing using engineered nucleases (meganucleases, zinc finger nucleases, transcription activator-like effector nucleases) has created many recent breakthroughs. Prescreening for efficiency and specificity is a critical step prior to using any newly designed genome editing tool for experimental purposes. The current standard screening methods of evaluation are based on DNA sequencing or use mismatch-sensitive endonucleases. They can be time-consuming and costly or lack reproducibility. Here, we review and critically compare standard techniques with those more recently developed in terms of reliability, time, cost, and ease of use.
RNA editing is a widespread co/posttranscriptional mechanism affecting primary RNAs by specific nucleotide modifications, which plays relevant roles in molecular processes including regulation of gene expression and/or the processing of noncoding RNAs. In recent years, the detection of editing sites has been improved through the availability of high-throughput RNA sequencing (RNA-Seq) technologies. Accurate bioinformatics pipelines are essential for the analysis of next-generation sequencing (NGS) data to ensure the correct identification of edited sites. Several pipelines, using various read mappers and variant callers with a wide range of adjustable parameters, are available for the detection of RNA editing events. In this review, we discuss some of the most recent and popular tools and provide guidelines for RNA-Seq data generation and analysis for the detection of RNA editing in massive transcriptome data. Using simulated and real data sets, we provide an overview of their behavior, emphasizing the fact that the RNA editing detection in NGS data sets remains a challenging task.
Genomic selection (GS) facilitates the rapid selection of superior genotypes and accelerates the breeding cycle. In this review, we discuss the history, principles, and basis of GS and genomic-enabled prediction (GP) as well as the genetics and statistical complexities of GP models, including genomic genotype×environment (G×E) interactions. We also examine the accuracy of GP models and methods for two cereal crops and two legume crops based on random cross-validation. GS applied to maize breeding has shown tangible genetic gains. Based on GP results, we speculate how GS in germplasm enhancement (i.e., prebreeding) programs could accelerate the flow of genes from gene bank accessions to elite lines. Recent advances in hyperspectral image technology could be combined with GS and pedigree-assisted breeding.
The CRISPR-Cas9 genome-editing system is a part of the adaptive immune system in archaea and bacteria to defend against invasive nucleic acids from phages and plasmids. The single guide RNA (sgRNA) of the system recognizes its target sequence in the genome, and the Cas9 nuclease of the system acts as a pair of scissors to cleave the double strands of DNA. Since its discovery, CRISPR-Cas9 has become the most robust platform for genome engineering in eukaryotic cells. Recently, the CRISPR-Cas9 system has triggered enormous interest in therapeutic applications. CRISPR-Cas9 can be applied to correct disease-causing gene mutations or engineer T cells for cancer immunotherapy. The first clinical trial using the CRISPR-Cas9 technology was conducted in 2016. Despite the great promise of the CRISPR-Cas9 technology, several challenges remain to be tackled before its successful applications for human patients. The greatest challenge is the safe and efficient delivery of the CRISPR-Cas9 genome-editing system to target cells in human body. In this review, we will introduce the molecular mechanism and different strategies to edit genes using the CRISPR-Cas9 system. We will then highlight the current systems that have been developed to deliver CRISPR-Cas9 in vitro and in vivo for various therapeutic purposes.
Regulation of cell proliferation is necessary for immune responses, tissue repair, and upkeep of organ function to maintain human health. When proliferating cells complete mitosis, a fraction of newly born daughter cells immediately enter the next cell cycle, while the remaining cells in the same population exit to a transient or persistent quiescent state. Whether this choice between two cell-cycle pathways is due to natural variability in mitogen signalling or other underlying causes is unknown. Here we show that human cells make this fundamental cell-cycle entry or exit decision based on competing memories of variable mitogen and stress signals. Rather than erasing their signalling history at cell-cycle checkpoints before mitosis, mother cells transmit DNA damage-induced p53 protein and mitogen-induced cyclin D1 (CCND1) mRNA to newly born daughter cells. After mitosis, the transferred CCND1 mRNA and p53 protein induce variable expression of cyclin D1 and the CDK inhibitor p21 that almost exclusively determines cell-cycle commitment in daughter cells. We find that stoichiometric inhibition of cyclin D1-CDK4 activity by p21 controls the retinoblastoma (Rb) and E2F transcription program in an ultrasensitive manner. Thus, daughter cells control the proliferation-quiescence decision by converting the memories of variable mitogen and stress signals into a competition between cyclin D1 and p21 expression. We propose a cell-cycle control principle based on natural variation, memory and competition that maximizes the health of growing cell populations.
Genomic evaluation has been successfully implemented in the United States, Canada, Great Britain, Ireland, New Zealand, Australia, France, the Netherlands, Germany, and the Scandinavian countries. Adoption of this technology in the major dairy producing countries has led to significant changes in the worldwide dairy industry. Gradual elimination of the progeny test system has led to a reduction in the number of sires with daughter records and fewer genetic ties between years. As genotyping costs decrease, the number of cows genotyped will continue to increase, and these records will become the basic data used to compute genomic evaluations, most likely via application of "single-step" methodologies. Although genomic selection has been successful in increasing rates of genetic gain, we still know very little about the genetic architecture of quantitative variation. Apparently, a very large number of genes affect nearly all economic traits, in accordance with the infinitesimal model for quantitative traits. Less emphasis in selection goals will be placed on milk production traits, and more on health, reproduction, and efficiency traits and on environmentally friendly production with reduced waste and gas emission. Genetic variance for economic traits is maintained by the increase in frequency of rare alleles, new mutations, and changes in selection goals and management. Thus, it is unlikely that a selection plateau will be reached in the near future.
Scientists are striving for a deeper view of development, from embryo to adult, cell-by-cell.
DNA of 234-year-old tree has few mutations, giving weight to idea that plants protect their stem cells.
Plant breeding has traditionally relied on combining the genetic diversity present within a species to develop combinations of alleles that provide desired traits. Epigenetic diversity may provide additional sources of variation within a species that could be captured or created for crop improvement. It will be important to understand the sources of epigenetic variation and the stability of newly formed epigenetic variants over generations to fully use the potential of epigenetic variation to improve crops. The development and application of methods for widespread epigenome profiling and engineering may generate new avenues for using the full potential of epigenetics in crop improvement.
The use of molecular signatures to add value to standard clinical and pathological parameters has impacted clinical practice in many cancer types, but perhaps most notably in the breast cancer field. This is, in part, due to the considerable complexity of the disease at the clinical, morphological and molecular levels. The adoption of molecular profiling of DNA, RNA and protein continues to reveal important differences in the intrinsic biology between molecular subtypes and has begun to impact the way patients are managed. Several bioinformatic tools have been developed using DNA or RNA-based signatures to stratify the disease into biologically and/or clinically meaningful subgroups. Here, we review the approaches that have been used to develop gene expression signatures into currently available diagnostic assays (e.g. OncotypeDX® and Mammaprint®), plus we describe the latest work on genome sequencing, the methodologies used in the discovery process of mutational signatures, and the potential of these signatures to impact the clinic.
Sequence alignment is an active research area in the field of bioinformatics. It is also a crucial task as it guides many other tasks like phylogenetic analysis, function, and/or structure prediction of biological macromolecules like DNA, RNA, and Protein. Proteins are the building blocks of every living organism. Although protein alignment problem has been studied for several decades, unfortunately, every available method produces alignment results differently for a single alignment problem. Multiple sequence alignment is characterized as a very high computational complex problem. Many stochastic methods, therefore, are considered for improving the accuracy of alignment. Among them, many researchers frequently use Genetic Algorithm. In this study, we have shown different types of the method applied in alignment and the recent trends in the multiobjective genetic algorithm for solving multiple sequence alignment. Many recent studies have demonstrated considerable progress in finding the alignment accuracy.
Because plants do not possess a proper germline, deleterious mutations that occur in the soma can be passed to gametes. It has generally been assumed that the large number of somatic cell divisions separating zygote from gamete formation in long-lived plants should lead to many mutations. However, a recent study showed that surprisingly few cell divisions separate apical stem cells from axillary stem cells in annual plants, challenging this view. To test this prediction, we generated and analysed the full genome sequence of two terminal branches of a 234-year-old oak tree and found very few fixed somatic single-nucleotide variants (SNVs), whose sequential appearance in the tree could reliably be traced back along nested sectors of younger branches. Our data indicate that the stem cells of shoot meristems in trees are robustly protected from accumulation of mutations, analogous to the germline in animals.
Single-cell genomic sequencing is poised to revolutionize fields from cancer to immunology.