Current risk assessment approaches for environmental and food and feed safety assessment.

Foundational activities at the international level underlie current risk and safety assessment approaches for genetically engineered/modified organisms (GEOs/GMOs). Early risk assessment considerations beginning with the OECD 'Blue Book' established risk/safety assessment as the characterization of the organism and its environmental release; establishment and persistence in the environment; and human and ecological effects, analyzed in principle through existing methods. Important in this context was recognition that GEOs/GMOs as a class did not represent new risks relative to products of traditional plant breeding and that any incremental risk would need to be established on a stepwise case-by-case comparative basis with existing crops and derived-foods as the baseline. Accordingly, concepts of familiarity and substantial equivalence were advanced by OECD and WHO as ways to establish a risk analysis baseline for determining whether and to what extent risk/safety assessment was needed. Regulatory implementations of this paradigm have skewed to increasingly complex portfolios of studies rather than adhering to analysis which is formulated to fit the risk/safety questions relevant to a given case. Plants produced through genome editing technology will benefit from risk analysis that implements sound problem formulation to guide the need for and nature of risk/safety assessments.

The Regulatory Status of Genome-edited Crops.

Genome editing with engineered nucleases (GEEN) represents a highly specific and efficient tool for crop improvement with the potential to rapidly generate useful novel phenotypes/traits. Genome editing techniques initiate specifically targeted double strand breaks facilitating DNA-repair pathways that lead to base additions or deletions by non-homologous end joining as well as targeted gene replacements or transgene insertions involving homology-directed repair mechanisms. Many of these techniques and the ancillary processes they employ generate phenotypic variation that is indistinguishable from that obtained through natural means or conventional mutagenesis; and therefore, they do not readily fit current definitions of genetically engineered or genetically modified used within most regulatory regimes. Addressing ambiguities regarding the regulatory status of genome editing techniques is critical to their application for development of economically useful crop traits. Continued regulatory focus on the process used, rather than the nature of the novel phenotype developed, results in confusion on the part of regulators, product developers, and the public alike and creates uncertainty as of the use of genome engineering tools for crop improvement.

Thermostability in endoglucanases is fold-specific.

BACKGROUND: Endoglucanases are usually considered to be synergistically involved in the initial stages of cellulose breakdown-an essential step in the bioprocessing of lignocellulosic plant materials into bioethanol. Despite their economic importance, we currently lack a basic understanding of how some endoglucanases can sustain their ability to function at elevated temperatures required for bioprocessing, while others cannot. In this study, we present a detailed comparative analysis of both thermophilic and mesophilic endoglucanases in order to gain insights into origins of thermostability. We analyzed the sequences and structures for sets of endoglucanase proteins drawn from the Carbohydrate-Active enZymes (CAZy) database. RESULTS: Our results demonstrate that thermophilic endoglucanases and their mesophilic counterparts differ significantly in their amino acid compositions. Strikingly, these compositional differences are specific to protein folds and enzyme families, and lead to differences in intramolecular interactions in a fold-dependent fashion. CONCLUSIONS: Here, we provide fold-specific guidelines to control thermostability in endoglucanases that will aid in making production of biofuels from plant biomass more efficient.

Problem formulation in the environmental risk assessment for genetically modified plants.

Problem formulation is the first step in environmental risk assessment (ERA) where policy goals, scope, assessment endpoints, and methodology are distilled to an explicitly stated problem and approach for analysis. The consistency and utility of ERAs for genetically modified (GM) plants can be improved through rigorous problem formulation (PF), producing an analysis plan that describes relevant exposure scenarios and the potential consequences of these scenarios. A properly executed PF assures the relevance of ERA outcomes for decision-making. Adopting a harmonized approach to problem formulation should bring about greater uniformity in the ERA process for GM plants among regulatory regimes globally. This paper is the product of an international expert group convened by the International Life Sciences Institute (ILSI) Research Foundation.

Prospective formulation of environmental risk assessments: probabilistic screening for Cry1A(b) maize risk to aquatic insects.

A critical first step for environmental risk assessment is problem formulation to identify environmental entities of concern and determinants of exposure that place these entities at risk. A conservative screening level approach was used to evaluate the potential risk to sensitive aquatic species from maize-expressed Cry1A(b) protein occurring in a representative agroecosystem. Estimated environmental concentrations for Cry1A(b) were compared to threshold concentrations of concern for putative sensitive aquatic organisms as estimated from species sensitivity distributions. The high-end risk expressed as the combined probability of short-term exposure and acute effects to a sensitive species indicated no concern in 99% of cases with limited opportunity for chronic effects due to the rapid decline of Cry1A(b) from the environment. Addressing uncertainties in the distribution of Cry1A(b) in soil, water, and sediment clarify the need for expanded ecotoxicity testing for aquatic effects.

Persistence and degradation of maize-expressed vaccine protein, Escherichia coli heat-labile enterotoxin subunit B, in soil and water.

Transgenic plants represent an innovative platform for the cost-effective large-scale production of various pharmaceutical proteins. The eventual open-field production of plant-made pharmaceuticals (PMPs) requires risk assessment to determine the potential for harm to the surrounding ecosystem. In the present study, the environmental persistence of a transgenic maize-expressed antigen, Escherichia coli heat-labile enterotoxin subunit B (LTB), was studied under laboratory conditions. To semiquantitatively monitor the persistence of LTB in soil, extraction with a high-salt, high-pH extraction buffer was optimized using the closely homologous Vibrio cholerae enterotoxin subunit B (CTB) as a test substance. The time to dissipation of 50% (DT50) of the extractable fraction of maize-expressed LTB was 4 to 15 d in pond water and 35 to 90 d in soils. Both extraction efficacy and persistence were strongly affected by the matrix type and incubation conditions. In contrast with maize-expressed LTB, the DT50 for bacterially produced LTB and CTB was less than 4 d both in pond water and soil. Although maize-expressed LTB was more stable than bacterially produced analogue, its dissipation was governed by an initial lag, which could be attributed to release from the plant material, followed by rapid decline.

Policy and Governance Perspectives for Regulation of Genome Edited Crops in the United States.

Genome editing for crop improvement lies at the leading edge of disruptive bioengineering technologies that will challenge existing regulatory paradigms for products of biotechnology and which will elicit widespread public interest. Regulation of products of biotechnology through the US Coordinated Framework for Biotechnology is predicated on requiring burden of proof that regulation is warranted. Although driven by considerations of newly emerging processes for product development, regulation has, for the most part, focused on characteristics of the biotechnology product itself and not the process used for its development per se. This standard of evidence and product focus has been maintained to date in regulatory considerations of genome edited crops. Those genome edited crops lacking recombinant DNA (rDNA) in the product intended for environmental release, lacking plant pest or pesticidal activity, or showing no food safety attributes different from those of traditionally bred crops are not deemed subject to regulatory evaluation. Regardless, societal uncertainties regarding genome editing are leading regulators to seek ways whereby these uncertainties may be addressed through redefinition of those products of biotechnology that may be subject to regulatory assessments. Within US law prior statutory history, language and regulatory action have significant influence on decision making; therefore, the administrative law and jurisprudence underlying the current Coordinated Framework strongly inform policy and governance when considering new plant breeding technologies such as genome editing.

Novel Features and Considerations for ERA and Regulation of Crops Produced by Genome Editing.

Genome editing describes a variety of molecular biology applications enabling targeted and precise alterations of the genomes of plants, animals and microorganisms. These rapidly developing techniques are likely to revolutionize the breeding of new crop varieties. Since genome editing can lead to the development of plants that could also have come into existence naturally or by conventional breeding techniques, there are strong arguments that these cases should not be classified as genetically modified organisms (GMOs) and be regulated no differently from conventionally bred crops. If a specific regulation would be regarded necessary, the application of genome editing for crop development may challenge risk assessment and post-market monitoring. In the session "Plant genome editing-any novel features to consider for ERA and regulation?" held at the 14th ISBGMO, scientists from various disciplines as well as regulators, risk assessors and potential users of the new technologies were brought together for a knowledge-based discussion to identify knowledge gaps and analyze scenarios for the introduction of genome-edited crops into the environment. It was aimed to enable an open exchange forum on the regulatory approaches, ethical aspects and decision-making considerations.

Low-dose exposure and immunogenicity of transgenic maize expressing the Escherichia coli heat-labile toxin B subunit.

BACKGROUND: Transgenic maize, which produces the nontoxic B subunit of the Escherichia coli heat-labile toxin (LT-B) in seed, has proven to be an effective oral immunogen in mice. Currently, there is considerable concern over accidental consumption of transgenic maize expressing LT-B by humans and domestic animals. We have yet to define nonimmunogenic levels of transgenic LT-B when ingested. OBJECTIVES: Our goal in this study was to determine the highest dose of LT-B orally administered in mice that does not result in a measurable immune response. We defined an immune response as specific serum or mucosal IgG or IgA significantly greater than background after three feedings (0.0002-20 mug) or a priming response induced by the intermittent feeding. METHODS: We fed transgenic maize pellets on days 0, 7, 21, and 49 and collected serum and fecal samples weekly. Serum was analyzed for LT-B-specific IgG and IgA, and feces was analyzed for LT-B-specific IgA. RESULTS: We observed a dose-dependent anti-LT-B antibody response with high specific antibody concentrations in groups fed high doses (0.2, 2, 20 mug) of LT-B maize. Mice fed 0.02 mug LT-B demonstrated immune priming in 62.5% of the animals. Mice that were fed

Safety, Security, and Policy Considerations for Plant Genome Editing.

Genome editing with engineered nucleases (GEEN) is increasingly used as a tool for gene discovery and trait development in crops through generation of targeted changes in endogenous genes. The development of the CRISPR-Cas9 system (clustered regularly interspaced short palindromic repeats with associated Cas9 protein), in particular, has enabled widespread use of genome editing. Research to date has not comprehensively addressed genome-editing specificity and off-target mismatches that may result in unintended changes within plant genomes or the potential for gene drive initiation. Governance and regulatory considerations for bioengineered crops derived from using GEEN will require greater clarity as to target specificity, the potential for mismatched edits, unanticipated downstream effects of off-target mutations, and assurance that genome reagents do not occur in finished products. Since governance and regulatory decision making involves robust standards of evidence extending from the laboratory to the postcommercial marketplace, developers of genome-edited crops must anticipate significant engagement and investment to address questions of regulators and civil society.

Risk associated with off-target plant genome editing and methods for its limitation.

Assessment for potential adverse effects of plant genome editing logically focuses on the specific characteristics of the derived phenotype and its release environment. Genome-edited crops, depending on the editing objective, can be classified as either indistinguishable from crops developed through conventional plant breeding or as crops which are transgenic. Therefore, existing regulatory regimes and risk assessment procedures accommodate genome-edited crops. The ability for regulators and the public to accept a product focus in the evaluation of genome-edited crops will depend on research which clarifies the precision of the genome-editing process and evaluates unanticipated off-target edits from the process. Interpretation of genome-wide effects of genome editing should adhere to existing frameworks for comparative risk assessment where the nature and degree of effects are considered relative to a baseline of genome-wide mutations as found in crop varieties developed through conventional breeding methods. Research addressing current uncertainties regarding unintended changes from plant genome editing, and adopting procedures that clearly avoid the potential for gene drive initiation, will help to clarify anticipated public and regulatory questions regarding risk of crops derived through genome editing.

Achieving Plant CRISPR Targeting that Limits Off-Target Effects.

The CRISPR-Cas9 system (clustered regularly interspaced short palindromic repeats with associated Cas9 protein) has been used to generate targeted changes for direct modification of endogenous genes in an increasing number of plant species; but development of plant genome editing has not yet fully considered potential off-target mismatches that may lead to unintended changes within the genome. Assessing the specificity of CRISPR-Cas9 for increasing editing efficiency as well as the potential for unanticipated downstream effects from off-target mutations is an important regulatory consideration for agricultural applications. Increasing genome-editing specificity entails developing improved design methods that better predict the prevalence of off-target mutations as a function of genome composition and design of the engineered ribonucleoprotein (RNP). Early results from CRISPR-Cas9 genome editing in plant systems indicate that the incidence of off-target mutation frequencies is quite low; however, by analyzing CRISPR-edited plant lines and improving both computational tools and reagent design, it may be possible to further decrease unanticipated effects at potential mismatch sites within the genome. This will provide assurance that CRISPR-Cas9 reagents can be designed and targeted with a high degree of specificity. Improved and experimentally validated design tools for discriminating target and potential off-target positions that incorporate consideration of the designed nuclease fidelity and selectivity will help to increase confidence for regulatory decision making for genome-edited plants.

Biomimetic extraction of Bacillus thuringiensis insecticidal crystal proteins from soil based on invertebrate gut fluid chemistry.

Quantitative monitoring of Bacillus thuringiensis (Bt) insecticidal crystal proteins in soil has been hampered by the lack of efficient extraction/detection methods. A novel approach for simple and effective Bt protein extraction was explored by evaluating extraction solutions from invertebrate gut fluids. Marine worm gut fluids were identified as promising for extracting Bt protein from soil. An artificial gut fluid based on these marine worm gut fluids was developed using commercially available chemicals and was evaluated for its ability to extract Bt proteins from soil. On the basis of experiments with Cry1 proteins, the artificial gut fluid in combination with ELISA was highly effective for protein extraction and analysis in a variety of soil types and was well-correlated with bioassay results. Coupling of immunoassay with this extraction method provides, for the first time, an efficient, accurate, and quantitative assay for routine measurement of Bt protein residues in soil.

Rapid degradation of the Cry1F insecticidal crystal protein in soil.

The gene for the core Cry1F insecticidal crystal protein (ICP) from Bacillus thuringiensis Berliner (Bt) has been incorporated into the genome of maize plants, Zea mays L. Plants expressing this ICP are protected from attack by various Lepidopteran pests including the European corn borer, Ostrinia nubilalis (Hübner). The stability of the Cry1F ICP in soil was assessed in a laboratory study designed to determine the persistence of the active protein residue in soil over time, using insect bioassay as the analytical quantification method. The GI(50) (concentration estimated to inhibit growth by 50%) rose at each consecutive incubation interval, indicating a consistent decline in Cry1F activity over time. The residue data were poorly described by a first-order model when fit to either the full data or a truncated data set where the last interval (28 days) was excluded. Data were well described by a shift-log model, and this model predicted DT(50) (time until 50% decay) and DT(90) (time until 90% decay) values of 0.6 and 6.9 days, respectively. This rapid degradation rate was consistent with other Bt proteins evaluated in our laboratory.

Activity and ecological implications of maize-expressed transgenic endo-1,4-ß-D-glucanase in agricultural soils.

Plant expression of thermostable endoglucanase (E1) has been proposed for improved conversion of lignocellulose to ethanol for fuel production. Residues of E1-expressing maize may affect ecological services (e.g., C mineralization and biogeochemical cycling) on soils where they occur. Therefore, the activity of residual E1 was investigated using soils amended with bacterial and plant-solubilized E1 compared with soil endogenous activity and residual activity from a mesostable cellulase (Aspergillus and Trichoderma spp.). An optimized analytical method involving a carboxymethyl cellulose substrate and dinitrosalicylic acid detection effectively assayed endoglucanase activity in amended and unamended soils and was used for determining E1 activity in 3 representative soils. The effect of E1 on soil carbon mineralization was determined by comparing CO(2) evolution from soils amended with transgenic E1-expressing and wild-type maize tissue. Extraction and recovery of the mesostable comparator, bacterial E1, and plant-soluble E1 showed nearly complete loss of exogenous endoglucanase activity within a 24-h period. Carbon mineralization indicated no significant difference between soils amended with either the transgenic E1 or wild-type maize tissue. These results indicate that maize residues expressing up to 30 µg E1/g tissue negligibly affect soil endoglucanase activity and CO(2) respiration for representative soils where transgenic E1 maize may be grown.

Africa's inevitable walk to genetically modified (GM) crops: opportunities and challenges for commercialization.

High relative poverty levels in Africa are attributed to the continent's under performing agriculture. Drought, low-yielding crop varieties, pests and diseases, poor soils, low fertilizer use, limited irrigation and lack of modern technologies are among the problems that plague African agriculture. Genetically modified (GM) crops may possess attributes that can help overcome some of these constraints, but have yet to be fully embraced in the mix of technology solutions for African agriculture. Cognizant of this, South Africa, Burkina Faso and Egypt are steadily growing GM crops on a commercial scale. Countries like Kenya, Nigeria, and Uganda are increasingly field-testing these crops with the view to commercialize them. These countries show strong government support for GM technology. Progress by these first adopter nations provides an insight as to how GM crops are increasingly being viewed as one of the ways in which the continent can invigorate the agriculture sector and achieve food security.

Endoglucanases: insights into thermostability for biofuel applications.

Obtaining bioethanol from cellulosic biomass involves numerous steps, among which the enzymatic conversion of the polymer to individual sugar units has been a main focus of the biotechnology industry. Among the cellulases that break down the polymeric cellulose are endoglucanases that act synergistically for subsequent hydrolytic reactions. The endoglucanases that have garnered relatively more attention are those that can withstand high temperatures, i.e., are thermostable. Although our understanding of thermostability in endoglucanases is incomplete, some molecular features that are responsible for increased thermostability have been recently identified. This review focuses on the investigations of endoglucanases and their implications for biofuel applications.

A mixture toxicity approach for environmental risk assessment of multiple insect resistance genes.

Multiple substance considerations applied to chemical mixtures in ecological risk assessments can be logically extended to nontarget organism (NTO) risk assessment for pyramided trait crops expressing multiple insect resistance genes. A case instance is developed that considers a two-protein pyramid of Cry1F and Cry1Ac synthetic proteins expressed in cotton (Gossypium hirsutum L.). A mixture toxicity approach was used to arrive at the aggregated multisubstance potentially affected fraction (msPAF) of NTOs that may be at risk from exposure to Cry1F + Cry1Ac cotton in representative-use environments. Development of the msPAF for putative susceptible NTOs considered laboratory toxicity data for Lepidoptera expressed in terms of additive mixture toxicity as well as data on in planta expression of the Cry1F and Cry1Ac proteins and their translation into environmental loads and exposure concentrations. The msPAF based on tier 1 estimated environmental concentrations (EECs) and toxicity to Lepidoptera species-used as surrogate data for adverse effects to a putative susceptible species-provided a highly conservative estimate of effects on beneficial species and therefore is a ready means to conduct screening-level NTO risk assessments for pyramided crops.

Fumonisin B1 toxicity in grower-finisher pigs: a comparative analysis of genetically engineered Bt corn and non-Bt corn by using quantitative dietary exposure assessment modeling.

In this study, we investigate the long-term exposure (20 weeks) to fumonisin B(1) (FB(1)) in grower-finisher pigs by conducting a quantitative exposure assessment (QEA). Our analytical approach involved both deterministic and semi-stochastic modeling for dietary comparative analyses of FB(1) exposures originating from genetically engineered Bacillus thuringiensis (Bt)-corn, conventional non-Bt corn and distiller's dried grains with solubles (DDGS) derived from Bt and/or non-Bt corn. Results from both deterministic and semi-stochastic demonstrated a distinct difference of FB(1) toxicity in feed between Bt corn and non-Bt corn. Semi-stochastic results predicted the lowest FB(1) exposure for Bt grain with a mean of 1.5 mg FB(1)/kg diet and the highest FB(1) exposure for a diet consisting of non-Bt grain and non-Bt DDGS with a mean of 7.87 mg FB(1)/kg diet; the chronic toxicological incipient level of concern is 1.0 mg of FB(1)/kg of diet. Deterministic results closely mirrored but tended to slightly under predict the mean result for the semi-stochastic analysis. This novel comparative QEA model reveals that diet scenarios where the source of grain is derived from Bt corn presents less potential to induce FB(1) toxicity than diets containing non-Bt corn.