Insights into maize genome editing via CRISPR/Cas9

Maize is an important crop for billions of people as food, feed, and industrial raw material. It is a prime driver of the global agricultural economy as well as the livelihoods of millions of farmers. Genetic interventions, such as breeding, hybridization and transgenesis have led to increased productivity of this crop in the last 100 years. The technique of genome editing is the latest advancement in genetics. Genome editing can be used for targeted deletions, additions, and corrections in the genome, all aimed at genetic enhancement of crops. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein 9 (CRISPR/Cas9) system is a recent genome editing technique that is considered simple, precise, robust and the most revolutionary. This review summarizes the current state of the art and predicts future directions in the use of the CRISPR/Cas9 tool in maize crop improvement.     

Genome editing technologies and their applications in crop improvement

Crop improvement is very essential to meet the increasing global food demands and enhance food nutrition. Conventional crop-breeding methods have certain limitations such as taking lot of time and resources, and causing biosafety concerns. These limitations could be overcome by the recently emerged-genome editing technologies that can precisely modify DNA sequences at the genomic level using sequence-specific nucleases (SSNs). Among the artificially engineered SSNs, the CRISPR/Cas9 is the most recently developed targeted genome modification system and seems to be more efficient, inexpensive, easy, user-friendly and rapidly adopted genome-editing tool. Large-scale genome editing has not only improved the yield and quality but also has enhanced the disease resistance ability in several model and other major crops. Increasing case studies suggest that genome editing is an efficient, precise and powerful technology that can accelerate basic and applied research towards crop improvement. In this review, we briefly overviewed the structure and mechanism of genome editing tools and then emphatically reviewed the advances in the application of genome editing tools for crop improvement, including the most recent case studies with CRISPR/Cpf1 and base-editing technologies. We have also discussed the future prospects towards the improvement of agronomic traits in crops.     

Genome Editing—Principles and Applications for Functional Genomics Research and Crop Improvement

Genome editing technologies are powerful tools for studying gene function and for crop improvement. The technologies rely on engineered endonucleases to generate double stranded breaks (DSBs) at target loci. The DSBs are repaired through the error-prone non-homologous end joining (NHEJ) and homology-directed repair (HDR) pathways in cells, resulting in mutations and sequence replacement, respectively. In the widely used CRISPR/Cas9 system, the endonuclease Cas9 is targeted by a CRISPR small RNA to DNA sequence of interest. In this review, we describe the four available types of genome editing tools, ZFN, TALEN, CRISPR/Cas9 and CRISPR/Cpf1, and show their applications in functional genomics research and precision molecular breeding of crops.     

CRISPR/Cas植物基因组编辑技术及其在玉米中的应用*

CRISPR/Cas 系统具有操作简单、效率高等优势,为植物功能基因研究和作物遗传改良提供了重要支撑。介绍了CRISPR/Cas植物基因组编辑技术的研究进展,并对CRISPR/Cas系统及其衍生技术进行了详细比较;结合案例综述了CRISPR/Cas9基因编辑技术在玉米产量、品质、抗逆性改良,以及雄性不育系创制和单倍体诱导等方面的应用;同时针对CRISPR/Cas系统未来需要迫切解决的一些问题进行了分析和展望。     

Plant genome editing: ever more precise and wide reaching

Genome-editing technologies consisting of targeted mutagenesis and gene targeting enable us to modify genes of interest rapidly and precisely. The discovery in 2012 of CRISPR/Cas9 systems and their development as sequence-specific nucleases has brought about a paradigm shift in biology. Initially, CRISPR/Cas9 was applied in targeted mutagenesis to knock out a target gene. Thereafter, advances in genome-editing technologies using CRISPR/Cas9 developed rapidly, with base editing systems for transition substitution using a combination of Cas9 nickase and either cytidine or adenosine deaminase being reported in 2016 and 2017, respectively, and later in 2021 bringing reports of transversion substitution using Cas9 nickase, cytidine deaminase and uracil DNA glycosylase. Moreover, technologies for gene targeting and prime editing systems using DNA or RNA as donors have also been developed in recent years. Besides these precise genome-editing strategies, reports of successful chromosome engineering using CRISPR/Cas9 have been published recently. The application of genome editing to crop breeding has advanced in parallel with the development of these technologies. Genome-editing enzymes can be introduced into plant cells, and there are now many examples of crop breeding using genome-editing technologies. At present, it is no exaggeration to say that we are now in a position to be able to modify a gene precisely and rearrange genomes and chromosomes in a predicted way. In this review, we introduce and discuss recent highlights in the field of precise gene editing, chromosome engineering and genome engineering technology in plants.     

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.     

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.

     

CRISPR/Cas‐based tools for the targeted control of plant viruses

Plant viruses are known to infect most economically important crops and pose a major threat to global food security. Currently, few resistant host phenotypes have been delineated, and while chemicals are used for crop protection against insect pests and bacterial or fungal diseases, these are inefficient against viral diseases. Genetic engineering emerged as a way of modifying the plant genome by introducing functional genes in plants to improve crop productivity under adverse environmental conditions. Recently, new breeding technologies, and in particular the exciting CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR‐associated proteins) technology, was shown to be a powerful alternative to engineer resistance against plant viruses, thus has great potential for reducing crop losses and improving plant productivity to directly contribute to food security. Indeed, it could circumvent the “Genetic modification” issues because it allows for genome editing without the integration of foreign DNA or RNA into the genome of the host plant, and it is simpler and more versatile than other new breeding technologies. In this review, we describe the predominant features of the major CRISPR/Cas systems and outline strategies for the delivery of CRISPR/Cas reagents to plant cells. We also provide an overview of recent advances that have engineered CRISPR/Cas‐based resistance against DNA and RNA viruses in plants through the targeted manipulation of either the viral genome or susceptibility factors of the host plant genome. Finally, we provide insight into the limitations and challenges that CRISPR/Cas technology currently faces and discuss a few alternative applications of the technology in virus research.     

CRISPR/Cas9:基因编辑的新时代

基因编辑技术是通过核酸内切酶对基因组DNA进行定向改造的技术,可以实现对特定DNA碱基的缺失、替换等,常用的四种基因编辑工具分别是:巨型核酸酶、锌指核酸酶、转录激活因子样效应物核酸酶以及CRISPR/Cas9系统.其中CRISPR/Cas9系统作为一种新型的基因组编辑技术具有组成简单、特异性好、切割效率高的优点.该文对...     

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: a promising way to exploit genetic variation in plants

Creation of variation in existing gene pool of crop plants is the foremost requirement in crop improvement programmes. Genome editing is a tool to produce knock out of target genes either by introduction of insertion or by deletion that disrupts the function of a specific gene. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system is the most recent addition to the toolbox of sequence-specific nucleases that includes ZFNs and TALENs. The CRISPR/Cas9 system allows targeted cleavage of genomic DNA guided by a small noncoding RNA, resulting in gene modifications by both non-homologous end joining and homology-directed repair mechanisms. Here, we present an overview of mechanisms of CRISPR, its potential roles in creating variation in germplasm and applications of this novel interference pathway in crop improvement. The availability of the CRISPR/Cas9 system holds promise in facilitating both forward and reverse genetics and will enhance research in crops that lack genetic resources.     

基于CRISPR/Cas系统的多重基因编辑与调控技术

规律成簇的间隔短回文重复序列(clustered regularly interspaced short palindromic repeats, CRISPR)及其相关Cas蛋白所构建的CRISPR/Cas系统是古细菌或细菌中特有的一种获得性免疫系统。研究人员将其开发成基因编辑工具之后,凭借其高效、精准和通用性强等优点迅速成为合成生物学领域的热门研究方向,在生命科学、生物工程技术、食品科学及农作物育种等多个领域引发了革命性的影响。目前基于CRISPR/Cas系统单基因编辑与调控技术日益完善,但在多重基因编辑和调控方面仍存在挑战。本文聚焦基于CRISPR/Cas系统的多重基因编辑与调控技术开发及应用,针对单个细胞内实现多位点基因编辑或调控和细胞群体内实现多位点基因编辑或调控技术,依据作用原理对其进行了系统总结和阐述,包括基于CRISPR/Cas系统的双链断裂、单链断裂以及多重基因调控技术等。这些工作丰富了多重基因编辑与调控的工具,为CRISPR/Cas系统在多领域的应用作出了贡献。     

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。     

CRISPR/Cas9技术在小麦育种中的应用进展

小麦（Triticum aestivum L.）是世界上主要的农作物之一,在粮食安全供应中发挥重要作用。在过去的几十年,由于小麦基因组复杂和遗传转化困难,导致小麦的基础和应用研究落后于其他谷类作物。2014年小麦基因组编辑取得了显著进展,进而促进了小麦生物技术的发展。综述了CRISPR/Cas9技术在小麦育种中的研究进展,简单介绍了CRISPR/Cas9基因编辑技术的发现、原理和优缺点,指出小麦基因编辑过程中农杆菌介导的遗传转化较粒子轰击法可降低转基因沉默频率,未来将成为基因编辑过程中主流的遗传转化方式;优化sgRNA的启动子、选择同源保守序列做为靶点可以提高基因编辑效率;新开发的碱基编辑器和prime editor需引入更多突变类型。展望了进一步提高小麦基因编辑效率和安全性的可行性,以期为未来小麦育种工作提供参考。