Targeted gene disruption by use of CRISPR/Cas9 ribonucleoprotein complexes in the water flea Daphnia pulex.

The microcrustacean Daphnia pulex is an important model for environmental, ecological, evolutionary and developmental genomics because its adaptive life history displays plasticity in response to environmental changes. Even though the whole-genome sequence is available and omics data have actively accumulated for this species, the available tools for analyzing gene function have thus far been limited to RNAi (RNA interference) and TALEN (the transcription activator-like effector nuclease) systems. The development of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) system is thus expected to further increase the genetic tractability of D. pulex and to advance the understanding of this species. In this study, we developed a genome editing system for D. pulex using CRISPR/Cas9 ribonucleoprotein complexes (Cas9 RNPs). We first assembled a CRISPR single-guide RNA (sgRNA) specific to the Distal-less gene (Dll), which encodes a homeodomain transcription factor essential for distal limb development in invertebrates and vertebrates. Then, we injected Cas9 RNPs into eggs and evaluated its activity in vivo by a T7 endonuclease I assay. Injected embryos showed defective formation of the second antenna and disordered development of appendages, and indel mutations were detected in Dll loci, indicating that this technique successfully knocked out the target gene.

A generic Transcriptomics Reporting Framework (TRF) for 'omics data processing and analysis.

A generic Transcriptomics Reporting Framework (TRF) is presented that lists parameters that should be reported in 'omics studies used in a regulatory context. The TRF encompasses the processes from transcriptome profiling from data generation to a processed list of differentially expressed genes (DEGs) ready for interpretation. Included within the TRF is a reference baseline analysis (RBA) that encompasses raw data selection; data normalisation; recognition of outliers; and statistical analysis. The TRF itself does not dictate the methodology for data processing, but deals with what should be reported. Its principles are also applicable to sequencing data and other 'omics. In contrast, the RBA specifies a simple data processing and analysis methodology that is designed to provide a comparison point for other approaches and is exemplified here by a case study. By providing transparency on the steps applied during 'omics data processing and analysis, the TRF will increase confidence processing of 'omics data, and regulatory use. Applicability of the TRF is ensured by its simplicity and generality. The TRF can be applied to all types of regulatory 'omics studies, and it can be executed using different commonly available software tools.

Framework for the quality assurance of 'omics technologies considering GLP requirements.

'Omics technologies are gaining importance to support regulatory toxicity studies. Prerequisites for performing 'omics studies considering GLP principles were discussed at the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) Workshop Applying 'omics technologies in Chemical Risk Assessment. A GLP environment comprises a standard operating procedure system, proper pre-planning and documentation, and inspections of independent quality assurance staff. To prevent uncontrolled data changes, the raw data obtained in the respective 'omics data recording systems have to be specifically defined. Further requirements include transparent and reproducible data processing steps, and safe data storage and archiving procedures. The software for data recording and processing should be validated, and data changes should be traceable or disabled. GLP-compliant quality assurance of 'omics technologies appears feasible for many GLP requirements. However, challenges include (i) defining, storing, and archiving the raw data; (ii) transparent descriptions of data processing steps; (iii) software validation; and (iv) ensuring complete reproducibility of final results with respect to raw data. Nevertheless, 'omics studies can be supported by quality measures (e.g., GLP principles) to ensure quality control, reproducibility and traceability of experiments. This enables regulators to use 'omics data in a fit-for-purpose context, which enhances their applicability for risk assessment.

Framework for the quantitative weight-of-evidence analysis of 'omics data for regulatory purposes.

A framework for the quantitative weight-of-evidence (QWoE) analysis of 'omics data for regulatory purposes is presented. The QWoE framework encompasses seven steps to evaluate 'omics data (also together with non-'omics data): (1) Hypothesis formulation, identification and weighting of lines of evidence (LoEs). LoEs conjoin different (types of) studies that are used to critically test the hypothesis. As an essential component of the QWoE framework, step 1 includes the development of templates for scoring sheets that predefine scoring criteria with scores of 0-4 to enable a quantitative determination of study quality and data relevance; (2) literature searches and categorisation of studies into the pre-defined LoEs; (3) and (4) quantitative assessment of study quality and data relevance using the respective pre-defined scoring sheets for each study; (5) evaluation of LoE-specific strength of evidence based upon the study quality and study relevance scores of the studies conjoined in the respective LoE; (6) integration of the strength of evidence from the individual LoEs to determine the overall strength of evidence; (7) characterisation of uncertainties and conclusion on the QWoE. To put the QWoE framework in practice, case studies are recommended to confirm the relevance of its different steps, or to adapt them as necessary.

Applying 'omics technologies in chemicals risk assessment: Report of an ECETOC workshop.

Prevailing knowledge gaps in linking specific molecular changes to apical outcomes and methodological uncertainties in the generation, storage, processing, and interpretation of 'omics data limit the application of 'omics technologies in regulatory toxicology. Against this background, the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) convened a workshop Applying 'omics technologies in chemicals risk assessment that is reported herein. Ahead of the workshop, multi-expert teams drafted frameworks on best practices for (i) a Good-Laboratory Practice-like context for collecting, storing and curating 'omics data; (ii) the processing of 'omics data; and (iii) weight-of-evidence approaches for integrating 'omics data. The workshop participants confirmed the relevance of these Frameworks to facilitate the regulatory applicability and use of 'omics data, and the workshop discussions provided input for their further elaboration. Additionally, the key objective (iv) to establish approaches to connect 'omics perturbations to phenotypic alterations was addressed. Generally, it was considered promising to strive to link gene expression changes and pathway perturbations to the phenotype by mapping them to specific adverse outcome pathways. While further work is necessary before gene expression changes can be used to establish safe levels of substance exposure, the ECETOC workshop provided important incentives towards achieving this goal.

Chemical and toxicological characterization of an unresolved complex mixture-rich biodegraded crude oil.

Chemical and toxicological characterization of unresolved complex mixtures in the water-soluble fraction of an artificially weathered Norwegian Sea crude oil was determined by a combination of chemical analysis and toxicity testing in fish in vitro bioassays. The water-soluble fraction of the crude oil was separated into 14 increasingly polar fractions by preparative high-pressure liquid chromatography. The in vitro toxicity (7-ethoxyresorufin O-deethylase activity, estrogenicity, and metabolic inhibition) of these fractions was characterized in a primary culture of liver cells (hepatocytes) from rainbow trout (Oncorhynchus mykiss). The main contributor to toxicity was one of the most polar fractions, accounting gravimetrically for more than 70% of the organic material in the water-soluble fraction and dominated by an unresolved complex mixture. Chemical analysis by gas chromatography-mass spectrometry and comprehensive two-dimensional gas chromatography-time of flight-mass spectrometry identified a large number of cyclic and aromatic sulfoxide compounds and low amounts of benzothiophenes (<0.1% of total mass) in this fraction. Commonly monitored toxic components of crude oil (e.g., naphthalenes, polycyclic aromatic hydrocarbons, and alkylated phenols) eluted in less polar fractions, characterized by somewhat lower toxicity. Normalization of in vitro responses to the mass in each fraction demonstrated a more even distribution of toxicity, indicating that toxicity in the individual fractions was related to the amount of material present. Although polar and nonpolar compounds contribute additively to crude oil toxicity, the water-soluble fraction was dominated by polar compounds because of their high aqueous solubility and the high oil-water loading. Under these conditions, the polar unresolved complex mixture-rich fraction might account for a large portion of crude oil toxicity because of its high abundance in the water-soluble fraction.

Chronic toxicity of 2,4,2',4'-tetrabromodiphenyl ether on the marine alga Skeletonema costatum and the crustacean Daphnia magna.

Polybrominated diphenyl ethers form a group of highly hydrophobic and persistent chemicals that are detected in various environmental matrices, such as soil, water, sediments, and biota. Although a variety of biological effects have been documented in vertebrates, the effects in invertebrates seem to be largely unknown. The objective of the present study was to determine the toxic effects of 2,4,2',4'-tetrabromodiphenyl ether (BDE 47) on the growth of the marine diatom Skeletonema costatum and on the parthogenetic reproduction and filtering activity of the freshwater crustacean Daphnia magna. The results showed that BDE 47 caused growth inhibition in S. costatum (no-observed-effect concentration [NOEC], 6.6 microg/L; median effect concentration, 70 microg/L; 95% confidence interval, 64-77 microg/L) and depressed the reproductive output of D. magna (NOEC, 14 microg/L). No effects were seen on the filtering rate of D. magna at any of the concentrations tested. Although sublethal toxicity was observed at low-microg/L levels, documented environmental water concentrations are many orders of magnitude lower, thus suggesting that BDE 47 is of minor risk to these organisms through direct water exposure.