Canadian regulatory aspects of gene editing technologies

The development of gene editing techniques, capable of producing plants and animals with new and improved traits, is revolutionizing the world of plant and animal breeding and rapidly advancing to commercial reality. However, from a regulatory standpoint the Government of Canada views gene editing as another tool that will join current methods used to develop desirable traits in plants and animals. This is because Canada focusses on the potential risk resulting from the novelty of the trait, or plant or animal product entering the Canadian environment or market place, rather than the process or method by which it was created. The Canadian Food Inspection Agency is responsible for the regulation of the environmental release of plants with novel traits, and novel livestock feeds, while Health Canada is responsible for the regulation of novel foods. Environment and Climate Change Canada, in partnership with Health Canada, regulates modified animals for entry into the environment. In all cases, these novel products may be the result of conventional breeding, mutagenesis, recombinant DNA techniques or other methods of plant or animal breeding such as gene editing. This novelty approach allows the Canadian regulatory system to efficiently adjust to any new developments in the science of plant and animal breeding and allows for risk-appropriate regulatory decisions. This approach encourages innovation while maintaining science-based regulatory expertise. Canadian regulators work cooperatively with proponents to determine if their gene editing-derived product meets the definition of a novel product, and whether it would be subject to a pre-market assessment. Therefore, Canada’s existing regulatory system is well positioned to accommodate any new innovations or technologies in plant or animal breeding, including gene editing.

     

Genome editing with the CRISPR‐Cas system: an art,ethics and global regulatory perspective

Over the last three decades, the development of new genome editing techniques, such as ODM, TALENs, ZFNs and the CRISPR‐Cas system, has led to significant progress in the field of plant and animal breeding. The CRISPR‐Cas system is the most versatile genome editing tool discovered in the history of molecular biology because it can be used to alter diverse genomes (e.g. genomes from both plants and animals) including human genomes with unprecedented ease, accuracy and high efficiency. The recent development and scope of CRISPR‐Cas system have raised new regulatory challenges around the world due to moral, ethical, safety and technical concerns associated with its applications in pre‐clinical and clinical research, biomedicine and agriculture. Here, we review the art, applications and potential risks of CRISPR‐Cas system in genome editing. We also highlight the patent and ethical issues of this technology along with regulatory frameworks established by various nations to regulate CRISPR‐Cas‐modified organisms/products.     

Genome editing for resistance against plant pests and pathogens

The conventional breeding of crops struggles to keep up with increasing food needs and ever-adapting pests and pathogens. Global climate changes have imposed another layer of complexity to biological systems, increasing the challenge to obtain improved crop cultivars. These dictate the development and application of novel technologies, like genome editing (GE), that assist targeted and fast breeding programs in crops, with enhanced resistance to pests and pathogens. GE does not require crossings, hence avoiding the introduction of undesirable traits through linkage in elite varieties, speeding up the whole breeding process. Additionally, GE technologies can improve plant protection by directly targeting plant susceptibility (S) genes or virulence factors of pests and pathogens, either through the direct edition of the pest genome or by adding the GE machinery to the plant genome or to microorganisms functioning as biocontrol agents (BCAs). Over the years, GE technology has been continuously evolving and more so with the development of CRISPR/Cas. Here we review the latest advancements of GE to improve plant protection, focusing on CRISPR/Cas-based genome edition of crops and pests and pathogens. We discuss how other technologies, such as host-induced gene silencing (HIGS) and the use of BCAs could benefit from CRISPR/Cas to accelerate the development of green strategies to promote a sustainable agriculture in the future.

     

Applications and potential of genome editing in crop improvement

Genome-editing tools provide advanced biotechnological techniques that enable the precise and efficient targeted modification of an organism’s genome. Genome-editing systems have been utilized in a wide variety of plant species to characterize gene functions and improve agricultural traits. We describe the current applications of genome editing in plants, focusing on its potential for crop improvement in terms of adaptation, resilience, and end-use. In addition, we review novel breakthroughs that are extending the potential of genome-edited crops and the possibilities of their commercialization. Future prospects for integrating this revolutionary technology with conventional and new-age crop breeding strategies are also discussed.     

Raising Climate-Resilient Crops: Journey From the Conventional Breeding to New Breeding Approaches

BackgroundIn order to meet the demands of the ever-increasing human population, it has become necessary to raise climate-resilient crops. Plant breeding, which involves crossing and selecting superior gene pools, has contributed tremendously towards achieving this goal during the past few decades. The relatively newer methods of crop improvement based on genetic engineering are relatively simple, and targets can be achieved in an expeditious manner. More recently emerged genome editing technique using CRISPR has raised strong hopes among plant scientists for precise integration of valuable traits and removal of undesirable ones.ConclusionGenome editing using Site-Specific Nucleases (SSNs) is a good alternative to the plant breeding and genetic engineering approaches as it can modify the genomes specifically and precisely at the target site in the host genome. Another added advantage of the genome editing approach is the simpler biosafety regulations that have been adopted by many countries for commercialization of the products thus generated. This review provides a critical assessment of the available methods for improving the stress tolerance in crop plants. Special emphasis has been given on genome editing approach in light of the diversity of tools, which are being discovered on an everyday basis and the practical applications of the same. This information will serve as a beginner’s guide to initiate the crop improvement programs as well as giving technical insight to the expert to plan the research strategically to tackle even multigenic traits in crop plants.     

Regulatory hurdles for genome editing: process- vs. product-based approaches in different regulatory contexts

Novel plant genome editing techniques call for an updated legislation regulating the use of plants produced by genetic engineering or genome editing, especially in the European Union. Established more than 25 years ago and based on a clear distinction between transgenic and conventionally bred plants, the current EU Directives fail to accommodate the new continuum between genetic engineering and conventional breeding. Despite the fact that the Directive 2001/18/EC contains both process- and product-related terms, it is commonly interpreted as a strictly process-based legislation. In view of several new emerging techniques which are closer to the conventional breeding than common genetic engineering, we argue that it should be actually interpreted more in relation to the resulting product. A legal guidance on how to define plants produced by exploring novel genome editing techniques in relation to the decade-old legislation is urgently needed, as private companies and public researchers are waiting impatiently with products and projects in the pipeline. We here outline the process in the EU to develop a legislation that properly matches the scientific progress. As the process is facing several hurdles, we also compare with existing frameworks in other countries and discuss ideas for an alternative regulatory system.     

Crop plants with improved culture and quality traits for food,feed and other uses

The large French research project GENIUS (2012–2019, https://www6.inra.genius-project_eng/) provides a good showcase of current genome editing techniques applied to crop plants. It addresses a large variety of agricultural species (rice, wheat, maize, tomato, potato, oilseed rape, poplar, apple and rose) together with some models (Arabidopsis, Brachypodium, Physcomitrella). Using targeted mutagenesis as its work horse, the project is limited to proof of concept under confined conditions. It mainly covers traits linked to crop culture, such as disease resistance to viruses and fungi, flowering time, plant architecture, tolerance to salinity and plant reproduction but also addresses traits improving the quality of agricultural products for industrial purposes. Examples include virus resistant tomato, early flowering apple and low-amylose starch potato. The wide range of traits illustrates the potential of genome editing towards a more sustainable agriculture through the reduction of pesticides and to the emergence of innovative bio-economy sectors based on custom tailored quality traits.

     

Problem formulation and phenotypic characterisation for the development of novel crops

Phenotypic characterisation provides important information about novel crops that helps their developers to make technical and commercial decisions. Phenotypic characterisation comprises two activities. Product characterisation checks that the novel crop has the qualities of a viable product—the intended traits have been introduced and work as expected, and no unintended changes have been made that will adversely affect the performance of the final product. Risk assessment evaluates whether the intended and unintended changes are likely to harm human health or the environment. Product characterisation follows the principles of problem formulation, namely that the characteristics required in the final product are defined and criteria to decide whether the novel crop will have these properties are set. The hypothesis that the novel crop meets the criteria are tested during product development. If the hypothesis is corroborated, development continues, and if the hypothesis is falsified, the product is redesigned or its development is halted. Risk assessment should follow the same principles. Criteria that indicate the crop poses unacceptable risk should be set, and the hypothesis that the crop does not possess those properties should be tested. However, risk assessment, particularly when considering unintended changes introduced by new plant breeding methods such as gene editing, often ignores these principles. Instead, phenotypic characterisation seeks to catalogue all unintended changes by profiling methods and then proceeds to work out whether any of the changes are important. This paper argues that profiling is an inefficient and ineffective method of phenotypic characterisation for risk assessment. It discusses reasons why profiling is favoured and corrects some misconceptions about problem formulation.

     

Perspectives for epigenetic editing in crops

Site-specific nucleases (SSNs) have drawn much attention in plant biotechnology due to their ability to drive precision mutagenesis, gene targeting or allele replacement. However, when devoid of its nuclease activity, the underlying DNA-binding activity of SSNs can be used to bring other protein functional domains close to specific genomic sites, thus expanding further the range of applications of the technology. In particular, the addition of functional domains encoding epigenetic effectors and chromatin modifiers to the CRISPR/Cas ribonucleoprotein complex opens the possibility to introduce targeted epigenomic modifications in plants in an easily programmable manner. Here we examine some of the most important agronomic traits known to be controlled epigenetically and review the best studied epigenetic catalytic effectors in plants, such as DNA methylases/demethylases or histone acetylases/deacetylases and their associated marks. We also review the most efficient strategies developed to date to functionalize Cas proteins with both catalytic and non-catalytic epigenetic effectors, and the ability of these domains to influence the expression of endogenous genes in a regulatable manner. Based on these new technical developments, we discuss the possibilities offered by epigenetic editing tools in plant biotechnology and their implications in crop breeding.

     

红枣活性成分及其生物活性研究进展

红枣中的活性成分以多糖、黄酮类、环核苷酸类、多酚类、五环三萜类、生物碱为主,具有抗氧化、免疫调节及抗肿瘤、保护肝脏、降血糖、抗炎等多种生物活性。本文综述了红枣中活性成分及生物活性的研究进展,并对红枣产业的发展进行展望,为红枣中活性成分的开发与利用提供科学依据。     

Application of genome editing in farm animals: cattle

Milk and meat from cattle and buffaloes contribute 45% of the global animal protein supply, followed by chickens (31%), and pigs (20%). In 2016, the global cattle population of 1.0 billion head produced 6.5 billion tons of cows’ milk, and 66 million tons of beef. In the past century, cattle breeding programs have greatly increased the yield per animal with a resultant decrease in the emissions intensity per unit of milk or beef, but this has not been true in all regions. Genome editing research in cattle to date has focused on disease resistance (e.g. tuberculosis), production (e.g. myostatin knockout; production of all-male offspring), elimination of allergens (e.g. beta-lactoglobulin knockout) and welfare (e.g. polled or hornlessness) traits. Modeling has revealed how the use of genome editing to introduce beneficial alleles into cattle breeds could maintain or even accelerate the rate of genetic gain accomplished by conventional breeding programs, and is a superior approach to the lengthy process of introgressing those same alleles from distant breeds. Genome editing could be used to precisely introduce useful alleles (e.g. heat tolerance, disease resistance) and haplotypes into native locally-adapted cattle breeds, thereby helping to improve their productivity. As with earlier genetic engineering approaches, whether breeders will be able to employ genome editing in cattle genetic improvement programs will very much depend upon global decisions around the regulatory framework and governance of genome editing for food animals.

     

Human health concerns with GM crops

Biotechnology was used in the first generation of so-called 'GM' crops to provide growers with complimentary and sometimes alternative crop management solutions to pesticides. Selected host genes or genes identified from other plants or non-plant sources are modified or transferred to a crop plant. The new or altered protein expression resulting from these modifications confer on the plant a desired physiological trait, such as resistance to particular herbicides or insect pests. Second generation modifications provide traits such as enhanced nutritional or health-promoting characteristics that are of benefit to consumers. The commonly raised concerns about possible implications for human health are: inherent toxicity of the novel gene and their products, the potential to express novel antigenic proteins or alter levels of existing protein allergens, the potential for unintended effects resulting from alterations of host metabolic pathways or over expression of inherently toxic or pharmacologically active substances and the potential for nutrient composition in the new food occur differing significantly from a conventional counterpart. Foods produced using biotechnology are subjected to far greater levels of scrutiny than foods produced by traditional plant breeding techniques. The accepted analytical, nutritional and toxicological methods employed to support this scrutiny and to assess and assure that a 'GM' food is a safe and nutritious as its 'non-GM' counterpart are discussed. The challenges associated with identifying unintended effects in whole GM foods and the promise new (proteomics/genomic) technologies offer opposite traditional toxicity testing paradigms are appraised.     

Oligonucleotide‐directed mutagenesis for precision gene editing

Differences in gene sequences, many of which are single nucleotide polymorphisms, underlie some of the most important traits in plants. With humanity facing significant challenges to increase global agricultural productivity, there is an urgent need to accelerate the development of these traits in plants. oligonucleotide‐directed mutagenesis (ODM), one of the many tools of Cibus’ Rapid Trait Development System ( RTDS ^?) technology, offers a rapid, precise and non‐transgenic breeding alternative for trait improvement in agriculture to address this urgent need. This review explores the application of ODM as a precision genome editing technology, with emphasis on using oligonucleotides to make targeted edits in plasmid, episomal and chromosomal DNA of bacterial, fungal, mammalian and plant systems. The process of employing ODM by way of RTDS technology has been improved in many ways by utilizing a fluorescence conversion system wherein a blue fluorescent protein (BFP) can be changed to a green fluorescent protein (GFP) by editing a single nucleotide of the BFP gene (CAC→TAC; H66 to Y66). For example, dependent on oligonucleotide length, applying oligonucleotide‐mediated technology to target the BFP transgene in Arabidopsis thaliana protoplasts resulted in up to 0.05% precisely edited GFP loci. Here, the development of traits in commercially relevant plant varieties to improve crop performance by genome editing technologies such as ODM, and by extension RTDS , is reviewed.     

碱基编辑系统研究最新进展及应用

CRISPR系统能够在基因组DNA中完成精准编辑,但依赖于细胞内的同源重组(Homologydirected recombination,HDR)修复途径,且效率极低。基于CRISPR/Cas9系统开发的碱基编辑技术(Base editing)通过将失去切割活性的核酸酶与不同碱基脱氨基酶融合,构建了两套碱基编辑系统(Baseeditors,BE):胞嘧啶碱基编辑器(Cytosine base editor,CBE)和腺嘌呤碱基编辑器(Adenine base editor,ABE)。这两类编辑器分别能够在不产生DNA双链断裂的前提下在基因靶位点完成CT (GA)或AG (TC)的替换,最终实现精准的碱基编辑。目前碱基编辑技术已经广泛应用于基因治疗、动物模型构建、精准动物育种和基因功能分析等领域,为基础和应用研究提供了强大的技术工具。文中概括了碱基编辑技术的研发过程、技术优势、应用现状、存在问题及改进策略,以期为相关领域的科研人员了解和使用碱基编辑系统提供参考。     

Exploring the potential of CRISPR/Cas genome editing for vegetable crop improvement: An overview of challenges and approaches

Vegetables provide many nutrients in the form of fiber, vitamins, and minerals, which make them an important part of our diet. Numerous biotic and abiotic stresses can affect crop growth, quality, and yield. Traditional and modern breeding strategies to improve plant traits are slow and resource intensive. Therefore, it is necessary to find new approaches for crop improvement. Clustered regularly interspaced short palindromic repeats/CRISPR associated 9 (CRISPR/Cas9) is a genome editing tool that can be used to modify targeted genes for desirable traits with greater efficiency and accuracy. By using CRISPR/Cas9 editing to precisely mutate key genes, it is possible to rapidly generate new germplasm resources for the promotion of important agronomic traits. This is made possible by the availability of whole genome sequencing data and information on the function of genes responsible for important traits. In addition, CRISPR/Cas9 systems have revolutionized agriculture, making genome editing more versatile. Currently, genome editing of vegetable crops is limited to a few vegetable varieties (tomato, sweet potato, potato, carrot, squash, eggplant, etc.) due to lack of regeneration protocols and sufficient genome sequencing data. In this article, we summarize recent studies on the application of CRISPR/Cas9 in improving vegetable trait development and the potential for future improvement.     

Plant susceptibility genes as a source for potyvirus resistance

Virus infection depends on the resources provided by the host plant. A number of host proteins that enable potyvirus infection have been identified. The genes encoding them are called susceptibility genes (S-genes). Loss-of-susceptibility type of resistance is based on S-gene modifications leading to incompatible host–virus interactions. An increasing number of examples show that this is a viable method for resistance breeding. While the recent advancements in genome editing and sequencing have remarkably reduced the technical limitations, we still need to tackle many biological challenges to be able to utilise S-genes for durable and broad range potyvirus resistance to their full extent. Many lessons on functional redundancy between gene family members and durability of the resistance have been learned by studying the naturally occurring recessive resistance based on the interplay between eukaryotic initiation factors eIF4E and eIFiso4E and viral protein genome-linked (VPg). Nevertheless, the outcomes of the S-gene modifications on resistance or any other characteristic of the host plant cannot be predicted. In addition to the genetic background of the host, also the properties of the viral factors affect the efficiency of the resistance and the emergence of resistance-breaking mutations. Many potyviral protein–protein interactions occur in multiprotein complexes. This suggests that the susceptibility factors may interact with viral proteins as a part of multifaceted protein–protein interaction networks. Rather than reviewing exhaustively the S-genes involved in potyvirus infection, my intention here is to discuss in the light of selected examples, the prospects and challenges of the use of potyviral S-genes in resistance breeding.     

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.