Plant breeding involving genetic engineering does not result in unacceptable unintended effects in rice relative to conventional cross-breeding.

Advancements in -omics techniques provide powerful tools to assess the potential effects in composition of a plant at the RNA, protein and metabolite levels. These technologies can thus be deployed to assess whether genetic engineering (GE) causes changes in plants that go beyond the changes introduced by conventional plant breeding. Here, we compare the extent of transcriptome and metabolome modification occurring in leaves of four GE rice lines expressing Bacillus thuringiensis genes developed by GE and seven rice lines developed by conventional cross-breeding. The results showed that both types of crop breeding methods can bring changes at transcriptomic and metabolic levels, but the differences were comparable between the two methods, and were less than those between conventional non-GE lines were. Metabolome profiling analysis found several new metabolites in GE rice lines when compared with the closest non-GE parental lines, but these compounds were also found in several of the conventionally bred rice lines. Functional analyses suggest that the differentially expressed genes and metabolites caused by both GE and conventional cross-breeding do not involve detrimental metabolic pathways. The study successfully employed RNA-sequencing and high-performance liquid chromatography mass spectrometry technology to assess the unintended changes in new rice varieties, and the results suggest that GE does not cause unintended effects that go beyond conventional cross-breeding in rice.

Linking CRISPR-Cas9 interference in cassava to the evolution of editing-resistant geminiviruses.

BACKGROUND: Geminiviruses cause damaging diseases in several important crop species. However, limited progress has been made in developing crop varieties resistant to these highly diverse DNA viruses. Recently, the bacterial CRISPR/Cas9 system has been transferred to plants to target and confer immunity to geminiviruses. In this study, we use CRISPR-Cas9 interference in the staple food crop cassava with the aim of engineering resistance to African cassava mosaic virus, a member of a widespread and important family (Geminiviridae) of plant-pathogenic DNA viruses. RESULTS: Our results show that the CRISPR system fails to confer effective resistance to the virus during glasshouse inoculations. Further, we find that between 33 and 48% of edited virus genomes evolve a conserved single-nucleotide mutation that confers resistance to CRISPR-Cas9 cleavage. We also find that in the model plant Nicotiana benthamiana the replication of the novel, mutant virus is dependent on the presence of the wild-type virus. CONCLUSIONS: Our study highlights the risks associated with CRISPR-Cas9 virus immunity in eukaryotes given that the mutagenic nature of the system generates viral escapes in a short time period. Our in-depth analysis of virus populations also represents a template for future studies analyzing virus escape from anti-viral CRISPR transgenics. This is especially important for informing regulation of such actively mutagenic applications of CRISPR-Cas9 technology in agriculture.

Risk-Only Assessment of Genetically Engineered Crops Is Risky.

The risks of not considering benefits in risk assessment are often overlooked. Risks are also often evaluated without consideration of the broader context. We discuss these two concepts in relation to genetically engineered (GE) crops. The health, environmental, and economic risks and benefits of GE crops are exemplified and presented in the context of modern agriculture. Misattribution of unique risks to GE crops are discussed. It is concluded that the scale of modern agriculture is its distinguishing characteristic and that the greater knowledge around GE crops allows for a more thorough characterization of risk. By considering the benefits and risks in the context of modern agriculture, society will be better served and benefits will be less likely to be forgone.

Next-generation biocontainment systems for engineered organisms.

The increasing use of engineered organisms for industrial, clinical, and environmental applications poses a growing risk of spreading hazardous biological entities into the environment. To address this biosafety issue, significant effort has been invested in creating ways to confine these organisms and transgenic materials. Emerging technologies in synthetic biology involving genetic circuit engineering, genome editing, and gene expression regulation have led to the development of novel biocontainment systems. In this perspective, we highlight recent advances in biocontainment and suggest a number of approaches for future development, which may be applied to overcome remaining challenges in safeguard implementation.

Concerns regarding 'off-target' activity of genome editing endonucleases.

Genome editing (GE) tools ensure targeted mutagenesis and sequence-specific modification in plants using a wide resource of customized endonucleases; namely, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), and the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated protein) system. Among these, in recent times CRISPR/Cas9 has been widely used in functional genomics and plant genetic modification. A significant concern in the application of GE tools is the occurrence of 'off-target' activity and induced mutations, which may impede functional analysis and gene activity studies. Moreover, the 'off-target' activity results in either not reported or unknown, difficult to detect, produce non-quantifiable cellular signaling and physiological effects. In the past few years, several experimental methods have been developed to identify undesired mutations and to curtail 'off-target' cleavage. Improvement in target specificity and minimizing 'off-target' activity will offer better applications of GE technology in plant biology and crop improvement.

2015 Evidence Analysis Library Systematic Review on Advanced Technology in Food Production.

In the late 20th century, plant breeders began using molecular biology techniques such as recombinant DNA, also known as genetic engineering, along with traditional cross-breeding. Ten plant and one animal food have been approved for commercialization in the United States. Today, foods and ingredients from genetically engineered (GE) crops are present throughout the food supply, which has led to varying levels of acceptance. Much discussion exists among consumers and health professionals about the believability of statements made regarding benefits or risks of GE foods. The aim of this systematic review was to examine the evidence on the association of consumption of GE foods and ingredients derived from them on human health, specifically allergenicity, food safety, pesticide consumption, nutrient adequacy, inflammation, and antibiotic resistance. An expert panel conducted a systematic review on advanced technology in food production. The 30 developed questions focused on effects of human consumption of GE foods and the effects of human consumption of foods containing pesticide residues on human health. Primary research published from 1994 to 2014 were identified using PubMed and Agricultural Online Access databases. Additional studies were identified by searching references of review articles. Twenty-one studies met the inclusion criteria. Relevant research addressed five of 30 questions. Four questions focused on food allergenicity, the fifth on nutrient adequacy, and all received a Grade III (limited/weak) rating. No human studies addressed 25 questions on the consumption of foods produced using genetic engineering technologies on gene translocation, cancer, food safety, phenotype expression, inflammation and inflammatory markers, or antibiotic resistance. These questions received a Grade V (grade not assignable). Evidence from human studies did not reveal an association between adverse health effects and consumption of foods produced using genetic engineering technologies. Although the number of available human studies is small, they support that there are no clear adverse health effects-as they relate to allergenicity and nutrient adequacy-associated with consumption of GE foods. The present systematic review is aligned with a recent report by the National Academy of Sciences that included human and animal research.

Ethical and Safety Issues of Stem Cell-Based Therapy.

Results obtained from completed and on-going clinical studies indicate huge therapeutic potential of stem cell-based therapy in the treatment of degenerative, autoimmune and genetic disorders. However, clinical application of stem cells raises numerous ethical and safety concerns. In this review, we provide an overview of the most important ethical issues in stem cell therapy, as a contribution to the controversial debate about their clinical usage in regenerative and transplantation medicine. We describe ethical challenges regarding human embryonic stem cell (hESC) research, emphasizing that ethical dilemma involving the destruction of a human embryo is a major factor that may have limited the development of hESC-based clinical therapies. With previous derivation of induced pluripotent stem cells (iPSCs) this problem has been overcome, however current perspectives regarding clinical translation of iPSCs still remain. Unlimited differentiation potential of iPSCs which can be used in human reproductive cloning, as a risk for generation of genetically engineered human embryos and human-animal chimeras, is major ethical issue, while undesired differentiation and malignant transformation are major safety issues. Although clinical application of mesenchymal stem cells (MSCs) has shown beneficial effects in the therapy of autoimmune and chronic inflammatory diseases, the ability to promote tumor growth and metastasis and overestimated therapeutic potential of MSCs still provide concerns for the field of regenerative medicine. This review offers stem cell scientists, clinicians and patient's useful information and could be used as a starting point for more in-depth analysis of ethical and safety issues related to clinical application of stem cells.

Arresting Evolution.

Evolution in the form of selective breeding has long been harnessed as a useful tool by humans. However, rapid evolution can also be a danger to our health and a stumbling block for biotechnology. Unwanted evolution can underlie the emergence of drug and pesticide resistance, cancer, and weeds. It makes live vaccines and engineered cells inherently unreliable and unpredictable, and therefore potentially unsafe. Yet, there are strategies that have been and can possibly be used to stop or slow many types of evolution. We review and classify existing population genetics-inspired methods for arresting evolution. Then, we discuss how genome editing techniques enable a radically new set of approaches to limit evolution.

Scalable Production of HPV16 L1 Protein and VLPs from Tobacco Leaves.

Cervical cancer is the most common malignancy among women particularly in developing countries, with human papillomavirus (HPV) 16 causing 50% of invasive cervical cancers. A plant-based HPV vaccine is an alternative to the currently available virus-like particle (VLP) vaccines, and would be much less expensive. We optimized methods to express HPV16 L1 protein and purify VLPs from tobacco (Nicotiana benthamiana) leaves transfected with the magnICON deconstructed viral vector expression system. L1 proteins were extracted from agro-infiltrated leaves using a series of pH and salt mediated buffers. Expression levels of L1 proteins and VLPs were verified by immunoblot and ELISA, which confirmed the presence of sequential and conformational epitopes, respectively. Among three constructs tested (16L1d22, TPL1d22, and TPL1F), TPL1F, containing a full-length L1 and chloroplast transit peptide, was best. Extraction of HPV16 L1 from leaf tissue was most efficient (> 2.5% of total soluble protein) with a low-salt phosphate buffer. VLPs were purified using both cesium chloride (CsCl) density gradient and size exclusion chromatography. Electron microscopy studies confirmed the presence of assembled forms of HPV16 L1 VLPs. Collectively; our results indicated that chloroplast-targeted transient expression in tobacco plants is promising for the production of a cheap, efficacious HPV16 L1 VLP vaccine. Studies are underway to develop plant VLPs for the production of a cervical cancer vaccine.

Transgenic proteins in agricultural biotechnology: The toxicology forum 40th annual summer meeting.

During the 40th Annual Meeting of The Toxicology Forum, the current and potential future science, regulations, and politics of agricultural biotechnology were presented and discussed. The range of current commercial crops and commercial crop traits related to transgenic proteins were reviewed and example crop traits discussed, including insecticidal resistance conferred by Bt proteins and the development of nutritionally enhanced food such as Golden Rice. The existing regulatory framework in the USA, with an emphasis on US FDA's role in evaluating the safety of genetically engineered crops under the regulatory umbrella of the FD&C Act was reviewed. Consideration was given to the polarized politics surrounding agricultural biotechnology, the rise of open access journals, and the influence of the internet and social media in shaping public opinion. Numerous questions related to misconceptions regarding current products and regulations were discussed, highlighting the need for more scientists to take an active role in public discourse to facilitate public acceptance and adoption of new technologies and to enable science-based regulations.

Biocontainment through reengineered genetic codes.

It's only unnatural: Reengineered genetic codes can achieve biocontainment by specifying different meanings for protein sequences in modified versus natural organisms; the synthetic bacterium discussed here is dependent on unnatural amino acids because its reengineered genetic code specifies unnatural amino acids in essential genes.

Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro-derived oocytes and embryos.

Targeted modification of the pig genome can be challenging. Recent applications of the CRISPR/Cas9 system hold promise for improving the efficacy of genome editing. When a designed CRISPR/Cas9 system targeting CD163 or CD1D was introduced into somatic cells, it was highly efficient in inducing mutations. When these mutated cells were used with somatic cell nuclear transfer, offspring with these modifications were created. When the CRISPR/Cas9 system was delivered into in vitro produced presumptive porcine zygotes, the system was effective in creating mutations in eGFP, CD163, and CD1D (100% targeting efficiency in blastocyst stage embryos); however, it also presented some embryo toxicity. We could also induce deletions in CD163 or CD1D by introducing two types of CRISPRs with Cas9. The system could also disrupt two genes, CD163 and eGFP, simultaneously when two CRISPRs targeting two genes with Cas9 were delivered into zygotes. Direct injection of CRISPR/Cas9 targeting CD163 or CD1D into zygotes resulted in piglets that have mutations on both alleles with only one CD1D pig having a mosaic genotype. We show here that the CRISPR/Cas9 system can be used by two methods. The system can be used to modify somatic cells followed by somatic cell nuclear transfer. System components can also be used in in vitro produced zygotes to generate pigs with specific genetic modifications.