Predicted multispecies unintended effects from outdoor genome editing.

CRISPR/Cas9, a potent genetic engineering tool widely adopted in agriculture, is capable of introducing new characteristics into plants on a large scale and without conventional breeding methods. Despite its remarkable efficiency, concerns have arisen regarding unintended consequences in uncontrolled environments. Our aim was to assess potential activity in organisms that could be exposed to genome editing in uncontrolled environments.

Specificity Testing for NGT PCR-Based Detection Methods in the Context of the EU GMO Regulations.

The term new genomic techniques (NGTs) is an umbrella term used to describe a variety of techniques that can alter the genetic material of an organism and that have emerged or have been developed since 2001, when the existing genetically modified organism (GMO) legislation was adopted. The analytical framework used to detect GMOs in Europe is an established single harmonized procedure that is mandatory for the authorization of GM food and feed, thus generating a reliable, transparent, and effective labeling scheme for GMO products. However, NGT products can challenge the implementation and enforcement of the current regulatory system in the EU, relating in particular to the detection of NGT products that contain no foreign genetic material.

Establishment of targeted mutagenesis in soybean protoplasts using CRISPR/Cas9 RNP delivery via electro-transfection.

The soybean ( L.) is an important crop with high agronomic value. The improvement of agronomic traits through gene editing techniques has broad application prospects in soybean.

Are null segregants new combinations of heritable material and should they be regulated?

Through genome editing and other techniques of gene technology, it is possible to create a class of organism called null segregants. These genetically modified organisms (GMOs) are products of gene technology but are argued to have no lingering vestige of the technology after the segregation of chromosomes or deletion of insertions. From that viewpoint regulations are redundant because any unique potential for the use of gene technology to cause harm has also been removed.

Unintended Genomic Outcomes in Current and Next Generation GM Techniques: A Systematic Review.

Classical genetic engineering and new genome editing techniques, especially the CRISPR/Cas technology, increase the possibilities for modifying the genetic material in organisms. These technologies have the potential to provide novel agricultural traits, including modified microorganisms and environmental applications. However, legitimate safety concerns arise from the unintended genetic modifications (GM) that have been reported as side-effects of such techniques.

A DNA-Free Editing Platform for Genetic Screens in Soybean CRISPR/Cas9 Ribonucleoprotein Delivery.

CRISPR/Cas9-based ribonucleoprotein (RNP)-mediated system has the property of minimizing the effects related to the unwanted introduction of vector DNA and random integration of recombinant DNA. Here, we describe a platform based on the direct delivery of Cas9 RNPs to soybean protoplasts for genetic screens in knockout gene-edited soybean lines without the transfection of DNA vectors. The platform is based on the isolation of soybean protoplasts and delivery of Cas RNP complex.

Proteomic Profile of Glyphosate-Resistant Soybean under Combined Herbicide and Drought Stress Conditions.

While some genetically modified (GM) plants have been targeted to confer tolerance to abiotic stressors, transgenes are impacted by abiotic stressors, causing adverse effects on plant physiology and yield. However, routine safety analyses do not assess the response of GM plants under different environmental stress conditions. In the context of climate change, the combination of abiotic stressors is a reality in agroecosystems.

PEG-Delivered CRISPR-Cas9 Ribonucleoproteins System for Gene-Editing Screening of Maize Protoplasts.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology allows the modification of DNA sequences in vivo at the location of interest. Although CRISPR-Cas9 can produce genomic changes that do not require DNA vector carriers, the use of transgenesis for the stable integration of DNA coding for gene-editing tools into plant genomes is still the most used approach. However, it can generate unintended transgenic integrations, while Cas9 prolonged-expression can increase cleavage at off-target sites.

Author Correction: An integrated multi-omics analysis of the NK603 Roundup-tolerant GM maize reveals metabolism disturbances caused by the transformation process.

A correction has been published and is appended to both the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Revisiting Risk Governance of GM Plants: The Need to Consider New and Emerging Gene-Editing Techniques.

New and emerging gene-editing techniques make it possible to target specific genes in species with greater speed and specificity than previously possible. Of major relevance for plant breeding, regulators and scientists are discussing how to regulate products developed using these gene-editing techniques. Such discussions include whether to adopt or adapt the current framework for GMO risk governance in evaluating the impacts of gene-edited plants, and derived products, on the environment, human and animal health and society.

Systematic miRNome profiling reveals differential microRNAs in transgenic maize metabolism.

Background: While some genetically modified organisms (GMOs) are created to produce new double-stranded RNA molecules (dsRNA), in others, such molecules may occur as an unintended effect of the genetic engineering process. Furthermore, GMOs might produce naturally occurring dsRNA molecules in higher or lower quantities than its non-transgenic counterpart. This study is the first to use high-throughput technology to characterize the miRNome of commercialized GM maize events and to investigate potential alterations in miRNA regulatory networks.

Reply to 'Comments on two recent publications on GM maize and Roundup'.

The opinion expressed by Eriksson and colleagues' fails to recognise that there are no standard experimental designs for academic investigations involving omics analyses of genetically modified crops and that the only valid comparator to determine the effect of the process of transgenesis is a near isogenic variety grown at the same time and location, as was the case in our investigation of NK603 maize. Eriksson does not acknowledge that the quality of the rat liver tissues in our chronic Roundup toxicity study has neither been questioned nor branded as unsuitable for further investigation. In addition, Eriksson fails to appreciate that the statistical methods we used to analyse the liver metabolomics dataset are recognised as appropriate as some of a number of approaches that can be taken.

Transgene flow in Mexican maize revisited: Socio-biological analysis across two contrasting farmer communities and seed management systems.

The flow of transgenes into landraces and wild relatives is an important biosafety concern. The case of transgene flow into local maize varieties in Mexico (the center of origin of maize) has been intensively debated over the past 15 years, including legal, political, and environmental disputes fanned by the existence of a significant scientific controversy over the methods used for the detection of transgenes. The use of diverse approaches and a lack of harmonized methods specific to the detection and monitoring of transgenes in landraces have generated both positive and negative results regarding contamination of Mexican maize with genetically modified material over the years.

Molecular responses of genetically modified maize to abiotic stresses as determined through proteomic and metabolomic analyses.

Some genetically modified (GM) plants have transgenes that confer tolerance to abiotic stressors. Meanwhile, other transgenes may interact with abiotic stressors, causing pleiotropic effects that will affect the plant physiology. Thus, physiological alteration might have an impact on the product safety.

An integrated multi-omics analysis of the NK603 Roundup-tolerant GM maize reveals metabolism disturbances caused by the transformation process.

Glyphosate tolerant genetically modified (GM) maize NK603 was assessed as 'substantially equivalent' to its isogenic counterpart by a nutrient composition analysis in order to be granted market approval. We have applied contemporary in depth molecular profiling methods of NK603 maize kernels (sprayed or unsprayed with Roundup) and the isogenic corn to reassess its substantial equivalence status. Proteome profiles of the maize kernels revealed alterations in the levels of enzymes of glycolysis and TCA cycle pathways, which were reflective of an imbalance in energy metabolism.

Levels of DNA methylation and transcript accumulation in leaves of transgenic maize varieties.

Background: Prior to their release in the environment, transgenic crops are examined for their health and environmental safety. In addition, transgene expression needs to be consistent in order to express the introduced trait (e.g.

Proteome of Plasmopara viticola-infected Vitis vinifera provides insights into grapevine Rpv1/Rpv3 pyramided resistance to downy mildew.

Unlabelled: Grapevine is one of the major fruit crops worldwide and requires phytochemical use due to susceptibility to numerous pests, including downy mildew. The pyramiding of previous identified QTL resistance regions allows selection of genotypes with combined resistance loci in order to build up sustainable resistance. This study investigates resistance response of pyramided plants containing Rpv1 and Rpv3 loci to Plasmopara viticola infection process.

Effect of stacking insecticidal cry and herbicide tolerance epsps transgenes on transgenic maize proteome.

Background: The safe use of stacked transgenic crops in agriculture requires their environmental and health risk assessment, through which unintended adverse effects are examined prior to their release in the environment. Molecular profiling techniques can be considered useful tools to address emerging biosafety gaps. Here we report the first results of a proteomic profiling coupled to transgene transcript expression analysis of a stacked commercial maize hybrid containing insecticidal and herbicide tolerant traits in comparison to the single event hybrids in the same genetic background.

Comparative proteomic analysis of genetically modified maize grown under different agroecosystems conditions in Brazil.

Background: Profiling technologies allow the simultaneous measurement and comparison of thousands of cell components without prior knowledge of their identity. In the present study, we used two-dimensional gel electrophoresis combined with mass spectrometry to evaluate protein expression of Brazilian genetically modified maize hybrid grown under different agroecosystems conditions. To this effect, leaf samples were subjected to comparative analysis using the near-isogenic non-GM hybrid as the comparator.

Comparative proteomic analysis of off-type and normal phenotype somatic plantlets derived from somatic embryos of Feijoa (Acca sellowiana (O. Berg) Burret).

Morphological disorders in a relevant portion of emerged somatic embryos have been a limiting factor in the true-to-type plantlet formation in Acca sellowiana. In this sense, the present study undertook a comparison between normal phenotype and off-type somatic plantlets protein profiles by means of the 2-D DIGE proteomics approach. Off-type and normal phenotype somatic plantlets obtained at 10 and 20 days conversion were evaluated.

A comparative evaluation of the regulation of GM crops or products containing dsRNA and suggested improvements to risk assessments.

Changing the nature, kind and quantity of particular regulatory-RNA molecules through genetic engineering can create biosafety risks. While some genetically modified organisms (GMOs) are intended to produce new regulatory-RNA molecules, these may also arise in other GMOs not intended to express them. To characterise, assess and then mitigate the potential adverse effects arising from changes to RNA requires changing current approaches to food or environmental risk assessments of GMOs.