Engineering plant virus resistance: from RNA silencing to genome editing strategies

Viral diseases severely affect crop yield and quality, thereby threatening global food security. Genetic improvement of plant virus resistance is essential for sustainable agriculture. In the last decades, several modern technologies were applied in plant antiviral engineering. Here we summarized breakthroughs of the two major antiviral strategies, RNA silencing and genome editing. RNA silencing strategy has been used in antiviral breeding for more than thirty years, and many crops engineered to stably express small RNAs targeting various viruses have been approved for commercial release. Genome editing technology has emerged in the past decade, especially CRISPR/Cas, which provides new methods for genetic improvement of plant virus resistance and accelerates resistance breeding. Finally, we discuss the potential of these technologies for breeding crops, and the challenges and solutions they may face in the future.     

CRISPR/Cas9技术在非模式植物中的应用进展

基因组编辑技术的出现对植物遗传育种及作物性状的改良产生了深远意义。CRISPR/Cas(clustered regularly interspaced short palindromic repeat)是由成簇规律间隔短回文重复序列及其关联蛋白组成的免疫系统,其作用是原核生物(40%细菌和90%古细菌)用来抵抗外源遗传物质(噬菌体和病毒)的入侵。该技术实现了对基因组中多个靶基因同时进行编辑,与前两代基因编辑技术:锌指核酶(ZFNs)和转录激活因子样效应物核酶(TALENs)相比更加简单、廉价、高效。目前CRISPR/Cas9基因编辑技术已在拟南芥(Arabidopsis thaliana)、烟草(Nicotiana benthamiana)、水稻(Oryza sativa)、小麦(Triticum aestivum)、玉米(Zea mays)、番茄(tomato)等模式植物和多数大作物中实现了定点基因组编辑,其应用范围不断地向各类植物扩展。但与模式植物和一些大作物相比,CRISPR/Cas9基因编辑技术在非模式植物,尤其在一些小作物的应用中存在如载体构建、靶点设计、脱靶检测、同源重组等问题有待进一步完善。该文对CRISPR/Cas9技术在非模式植物与小作物研究的最新研究进展进行了总结,讨论了该技术目前在非模式植物、小作物应用的局限性,在此基础上提出了相关改进策略,并对CRISPR/Cas9系统在非模式植物中的研究前景进行了展望。     

Prospects for application of breakthrough technologies in breeding: The CRISPR/Cas9 system for plant genome editing

Integration of the methods of contemporary genetics and biotechnology into the breeding process is assessed, and the potential role and efficacy of genome editing as a novel approach is discussed. Use of molecular (DNA) markers for breeding was proposed more than 30 years ago. Nowadays, they are widely used as an accessory tool in order to select plants by mono- and olygogenic traits. Presently, the genomic approaches are actively introduced into the breeding processes owing to automatization of DNA polymorphism analyses and development of comparatively cheap methods of DNA sequencing. These approaches provide effective selection by complex quantitative traits, and are based on the full-genome genotyping of the breeding material. Moreover, biotechnological tools, such as doubled haploids production, which provides fast obtainment of homozygotes, are widely used in plant breeding. Use of genomic and biotechnological approaches makes the development of varieties less time consuming. It also decreases the cultivated areas and financial expenditures required for accomplishment of the breeding process. However, the capacities of modern breeding are not limited to only these advantages. Experiments carried out on plants about 10 years ago provided the first data on genome editing. In the last two years, we have observed a sharp increase in the number of publications that report about successful experiments aimed at plant genome editing owing to the use of the relatively simple and convenient CRISPR/Cas9 system. The goal of some of these experiments was to modify agriculturally valuable genes of cultivated plants, such as potato, cabbage, tomato, maize, rice, wheat, barley, soybean and sorghum. These studies show that it is possible to obtain nontransgenic plants carrying stably inherited, specifically determined mutations using the CRISPR/Cas9 system. This possibility offers the challenge to obtain varieties with predetermined mono- and olygogenic traits.     

Nuclear and plastid genetic engineering of plants: Comparison of opportunities and challenges

Plant genetic engineering is one of the key technologies for crop improvement as well as an emerging approach for producing recombinant proteins in plants. Both plant nuclear and plastid genomes can be genetically modified, yet fundamental functional differences between the eukaryotic genome of the plant cell nucleus and the prokaryotic-like genome of the plastid will have an impact on key characteristics of the resulting transgenic organism. So, which genome, nuclear or plastid, to transform for the desired transgenic phenotype? In this review we compare the advantages and drawbacks of engineering plant nuclear and plastid genomes to generate transgenic plants with the traits of interest, and evaluate the pros and cons of their use for different biotechnology and basic research applications, ranging from generation of commercial crops with valuable new phenotypes to ‘bioreactor’ plants for large-scale production of recombinant proteins to research model plants expressing various reporter proteins.     

植物基因组编辑检测方法

以CRISPR/Cas9技术为代表的基因组编辑在生物领域的革命性应用使得生命科学研究迈入新篇章。该技术以其灵活性、易用性且扩展性强等优势,大大加快了基因工程研究,也加速了植物分子育种的步伐。但是,遗传转化过程中产生大量潜在的基因编辑植株,使得早期高通量快速筛选和检测目标编辑植株面临很大挑战。本文综述了近年来植物基因组编辑检测的各种方法,比较了其优缺点和适用范围;同时,还对近几年植物基因组编辑检测方法的发展趋势进行了深入分析和展望,以期对基因组编辑技术在植物中的应用提供参考。     

Where are we with unintended effects in genome editing applications from DNA to phenotype: focus on plant applications

Agriculture has benefited from various conventional techniques for plant breeding, including chemical- or radiation-induced mutagenesis, and to some extent from transgenesis. Genome editing techniques are likely to allow straightforward, cost-effective and efficient gene-specific modifications for identified genetic traits associated to agronomic interest. As for previous plant breeding techniques, genome editing techniques need an appraisal for unintended effects. Hence, an evaluation of potential specific risks associated with genome editing must be considered. The Scientific Committee of the High Council for biotechnology (HCB), using a broad theoretical and literature-based approach, identified three categories of points to consider in terms of hazards in health and environment, as compared to conventional breeding: (1) technical unintended effects related to effector persistence as well as risks associated with off-target modifications or other unintended genome modifications, (2) risks arising from the desired trait and its novelty in the plant, and (3) risks associated with the potential modification of plant breeding practices, owing to efficacy and technical ease-of-use of genome editing (acceleration), be it for single traits or for combined modifications (multiplex genome editing). Due to novelty, HCB also envisions the need for specific risk assessment and management.

     

The Current Status of Culture Collections and Their Contribution to Biotechnology

Abstract

Cereals are the most important group of plants for human nutrition and animal feed. Partially due to the commercial value of crop plants, there has been an ever-increasing interest in using modern biotechnological methods for the improvement of the characteristics of cereals during the past decade. The rapid progress in molecular biology, plant cell culture techniques, and gene transfer technology has resulted in successful transformations of all the major cereals—maize, rice, wheat, and barley. This brings the biotechnological methods closer to the routine also in barley breeding. In this article, the current status of barley genetic engineering, including the patent situation, is reviewed. The needs, aims, and possible applications of genetic engineering in barley breeding are discussed.     

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.     

高等植物叶绿体基因组转化的应用

叶绿体基因组转化技术由于其独特的优越性,现已成为植物基因工程的研究热点。本文简单介绍了叶绿体基因组转化技术的原理和方法;并重点综述了该技术在基础研究和实践中的应用。这些应用主要包括利用叶绿体基因组转化技术进行Rubisco的组装,叶绿体基因结构、转录、翻译和RNA编辑等研究;利用叶绿体作为生物反应器生产人生长激素、霍乱毒素抗体、聚羟基丁酸脂和生物弹性蛋白等;获得抗虫、抗病、抗除草剂和耐旱的转基因植物;以及降低转基因植物的外源基因扩散等。     

Base editing in crops: current advances,limitations and future implications

Targeted mutagenesis via genome‐editing technologies holds great promise in developing improved crop varieties to meet future demands. Point mutations or single nucleotide polymorphisms often determine important agronomic traits of crops. Genome‐editing‐based single‐base changes could generate elite trait variants in crop plants which help in accelerating crop improvement. Among the genome‐editing technologies, base editing has emerged as a novel and efficient genome‐editing approach which enables direct and irreversible conversion of one target base into another in a programmable manner. A base editor is a fusion of catalytically inactive CRISPR–Cas9 domain (Cas9 variants) and cytosine or adenosine deaminase domain that introduces desired point mutations in the target region enabling precise editing of genomes. In the present review, we have summarized the development of different base‐editing platforms. Then, we have focussed on the current advances and the potential applications of this precise technology in crop improvement. The review also sheds light on the limitations associated with this technology. Finally, the future perspectives of this emerging technology towards crop improvement have been highlighted.     

TILLING by Sequencing (TbyS) for targeted genome mutagenesis in crops

TILLING (Targeting Induced Local Lesions in Genomes) by Sequencing (TbyS) refers to the application of high-throughput sequencing technologies to mutagenised TILLING populations as a tool for functional genomics. TbyS can be used to identify and characterise induced variation in genes (controlling traits of interest) within large mutant populations, and is a powerful approach for the study and harnessing of genetic variation in crop breeding programmes. The extension of existing TILLING platforms by TbyS will accelerate crop functional genomics studies, in concert with the rapid increase in genome editing capabilities and the number and quality of sequenced crop plant genomes. In this mini-review, we provide an overview of the growth of TbyS and its potential applications to crop molecular breeding.     

Biotechnology: Genetic improvement of sorghum (Sorghum bicolor (L.) Moench)

Summary This report reviews the contributions to the improvement of sorghum (Sorghum bicolor (L.) Moench) through traditional approaches with emphasis on the application of biotechnological methods. Strategies include breeding for higher yield, improved grain quality, and biotic and abiotic stress tolerance. Hybrid development and polyploidy breeding are also discussed. Plant breeders, working in concert with biotechnologists, have developed new powerful tools for plant genetic manipulation and genotype evaluation that will significantly improve the efficiency of plant breeding. Improving sorghum through biotechnology is the latest in a long series of technologies that have been applied to this crop. Five basic tools of technology have been developed for sorghum improvement: (1) in vitro protocols for efficient plant regeneration; (2) molecular markers; (3) gene identification and cloning; (4) genetic engineering and gene transfer technology to integrate desirable traits into the sorghum genome; and (5) genomics and germplasm databases. Reports on studies involving the problems, progress, and prospects for utilizing the biotechnological methods for sorghum improvement are discussed.     

CRISPR-Cas系统在植物基因组编辑中的研究进展

基因组定点编辑技术是研究基因功能和生物体改造的重要工具。CRISPR-Cas(Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins)系统是近年来发展的一种新型基因组编辑技术,该技术通过一段向导RNA和配套的核酸酶就可对特定的基因组序列进行定点编辑,具有简单高效、应用广泛的特点,受到了生物学家的广泛关注。本文着重介绍CRISPR-Cas系统在植物中的研究进展,包括CRISPR-Cas9系统在植物中的应用与完善、扩大基因组编辑范围的研究、Cas9切口酶和失活酶的拓展、特异性单碱基突变编辑系统的研究、无外源DNA污染的植物基因编辑技术的发展以及基因组编辑技术在作物育种上的应用等方面。同时也提出了还需解决的问题,并展望了基因组编辑系统在作物育种中的应用前景,为开展这一领域的研究工作提供参考。     

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.     

Current and future editing reagent delivery systems for plant genome editing

Many genome editing tools have been developed and new ones are anticipated; some have been extensively applied in plant genetics, biotechnology and breeding, especially the CRISPR/Cas9 system. These technologies have opened up a new era for crop improvement due to their precise editing of user-specified sequences related to agronomic traits. In this review, we will focus on an update of recent developments in the methodologies of editing reagent delivery, and consider the pros and cons of current delivery systems. Finally, we will reflect on possible future directions.     

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.