植物CRISPR基因组编辑技术的新进展

在过去的4年中,CRISPR/Cas9基因组编辑技术成为生命科学领域的革命性工具,为植物学基础研究和农作物遗传改良提供了高效、快速而又廉价的遗传操作工具。利用CRISPR/Cas9系统可以实现精准的knock-out和knock-in等遗传操作,也可用于靶向激活或抑制基因的表达。在CRISPR/Cas9被广泛地用于基因组编辑的同时,它的编辑能力、效率和精确度也在不断地改进和完善,特别是CRISPR/Cpf1系统的发掘和单碱基编辑技术的创建,使CRISPR系统正逐步成为一个理想的遗传工程技术平台。此外,利用CRISPR/Cas9技术改良的农作物品种也已经涌现,这必将推动精准基因组编辑技术在农作物遗传改良中的应用和发展。     

Rapid generation of genetic diversity by multiplex CRISPR/Cas9 genome editing in rice

The clustered regularly interspaced short palindromic repeats(CRISPR)-associated endonuclease 9(CRISPR/Cas9) system has emerged as a promising technology for specific genome editing in many species. Here we constructed one vector targeting eight agronomic genes in rice using the CRISPR/Cas9 multiplex genome editing system. By subsequent genetic transformation and DNA sequencing, we found that the eight target genes have high mutation efficiencies in the T_0 generation. Both heterozygous and homozygous mutations of all editing genes were obtained in T_0 plants. In addition, homozygous sextuple, septuple, and octuple mutants were identified. As the abundant genotypes in T_0 transgenic plants, various phenotypes related to the editing genes were observed. The findings demonstrate the potential of the CRISPR/Cas9 system for rapid introduction of genetic diversity during crop breeding.     

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.     

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.     

Optimized paired‐sgRNA/Cas9 cloning and expression cassette triggers high‐efficiency multiplex genome editing in kiwifruit

Kiwifruit is an important fruit crop; however, technologies for its functional genomic and molecular improvement are limited. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability is variable depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Optimizing conditions for its use within a particular species is therefore needed to achieve highly efficient genome editing. In this study, we developed a new cloning strategy for generating paired‐sgRNA/Cas9 vectors containing four sgRNAs targeting the kiwifruit phytoene desaturase gene (AcPDS). Comparing to the previous method of paired‐sgRNA cloning, our strategy only requires the synthesis of two gRNA‐containing primers which largely reduces the cost. We further compared efficiencies of paired‐sgRNA/Cas9 vectors containing different sgRNA expression devices, including both the polycistronic tRNA‐sgRNA cassette (PTG) and the traditional CRISPR expression cassette. We found the mutagenesis frequency of the PTG/Cas9 system was 10‐fold higher than that of the CRISPR/Cas9 system, coinciding with the relative expressions of sgRNAs in two different expression cassettes. In particular, we identified large chromosomal fragment deletions induced by the paired‐sgRNAs of the PTG/Cas9 system. Finally, as expected, we found both systems can successfully induce the albino phenotype of kiwifruit plantlets regenerated from the G418‐resistance callus lines. We conclude that the PTG/Cas9 system is a more powerful system than the traditional CRISPR/Cas9 system for kiwifruit genome editing, which provides valuable clues for optimizing CRISPR/Cas9 editing system in other plants.     

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。     

Trait discovery and editing in tomato

Tomato (Solanum lycopersicum), which is used for both processing and fresh markets, is a major crop species that is the top ranked vegetable produced over the world. Tomato is also a model species for research in genetics, fruit development and disease resistance. Genetic resources available in public repositories comprise the 12 wild related species and thousands of landraces, modern cultivars and mutants. In addition, high quality genome sequences are available for cultivated tomato and for several wild relatives, hundreds of accessions have been sequenced, and databases gathering sequence data together with genetic and phenotypic data are accessible to the tomato community. Major breeding goals are productivity, resistance to biotic and abiotic stresses, and fruit sensorial and nutritional quality. New traits, including resistance to various biotic and abiotic stresses and root architecture, are increasingly being studied. Several major mutations and quantitative trait loci (QTLs) underlying traits of interest in tomato have been uncovered to date and, thanks to new populations and advances in sequencing technologies, the pace of trait discovery has considerably accelerated. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing (GE) already proved its remarkable efficiency in tomato for engineering favorable alleles and for creating new genetic diversity by gene disruption, gene replacement, and precise base editing. Here, we provide insight into the major tomato traits and underlying causal genetic variations discovered so far and review the existing genetic resources and most recent strategies for trait discovery in tomato. Furthermore, we explore the opportunities offered by CRISPR/Cas9 and their exploitation for trait editing in tomato.     

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。     

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.     

The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation

The CRISPR/Cas9 system has been demonstrated to efficiently induce targeted gene editing in a variety of organisms including plants. Recent work showed that CRISPR/Cas9‐induced gene mutations in Arabidopsis were mostly somatic mutations in the early generation, although some mutations could be stably inherited in later generations. However, it remains unclear whether this system will work similarly in crops such as rice. In this study, we tested in two rice subspecies 11 target genes for their amenability to CRISPR/Cas9‐induced editing and determined the patterns, specificity and heritability of the gene modifications. Analysis of the genotypes and frequency of edited genes in the first generation of transformed plants (T0) showed that the CRISPR/Cas9 system was highly efficient in rice, with target genes edited in nearly half of the transformed embryogenic cells before their first cell division. Homozygotes of edited target genes were readily found in T0 plants. The gene mutations were passed to the next generation (T1) following classic Mendelian law, without any detectable new mutation or reversion. Even with extensive searches including whole genome resequencing, we could not find any evidence of large‐scale off‐targeting in rice for any of the many targets tested in this study. By specifically sequencing the putative off‐target sites of a large number of T0 plants, low‐frequency mutations were found in only one off‐target site where the sequence had 1‐bp difference from the intended target. Overall, the data in this study point to the CRISPR/Cas9 system being a powerful tool in crop genome engineering.     

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.     

Application and future perspective of CRISPR/Cas9 genome editing in fruit crops

Fruit crops, including apple, orange, grape,banana, strawberry, watermelon, kiwifruit and tomato, not only provide essential nutrients for human life but also contribute to the major agricultural output and economic growth of many countries and regions in the world. Recent advancements in genome editing provides an unprecedented opportunity for the genetic improvement of these agronomically important fruit crops. Here, we summarize recent reports of applying CRISPR/Cas9 to fruit crops,including efforts to reduce disease susceptibility, change plant architecture or flower morphology, improve fruit quality traits, and increase fruit yield. We discuss challenges facing fruit crops as well as new improvements and platforms that could be used to facilitate genome editing in fruit crops, including d Cas9-base-editing to introduce desirable alleles and heat treatment to increase editing efficiency. In addition, we highlight what we see as potentially revolutionary development ranging from transgene-free genome editing to de novo domestication of wild relatives. Without doubt, we now see only the beginning of what will eventually be possible with the use of the CRISPR/Cas9 toolkit. Efforts to communicate with the public and an emphasis on the manipulation of consumerfriendly traits will be critical to facilitate public acceptance of genetically engineered fruits with this new technology.     

CRISPR/Cas9系统在昆虫中的应用

CRISPR/Cas9(clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9)技术是一种RNA引导的基因组靶向编辑技术,能对基因组序列进行精确编辑,在探究基因功能、修复受损基因、沉默有害基因、改良品质性状等方面具有广阔的应用前景。近年来,随着对CRISPR/Cas9系统研究的不断深入和改造,该系统以其操作简易、省时、高效等优点在生物学研究的众多领域中得以推广和应用,特别是在果蝇(Bombyx mori)、家蚕(silkworm)、埃及伊蚊(Aedes aegypti)和蝴蝶(butterfly)等多种昆虫中。本文概述了CRISPR/Cas9的结构、作用原理及发展优化,总结了CRISPR/Cas9导入昆虫的策略和在昆虫中的应用,以及对CRISPR/Cas9系统产生脱靶问题的应对策略,以期对经济昆虫和有益昆虫的分子育种、害虫的生物技术防控等研究提供参考。     

基因组编辑技术在猪遗传改良中的应用

核酸酶介导的基因组编辑技术大幅度提高了编辑真核细胞基因组的能力,给生命科学领域带来了革命性地发展,也给猪的遗传改良带来了全新的契机。本文介绍了基因组编辑技术尤其是CRISPR/Cas9系统的发展以及各种天然存在的和人为改造的Cas9变体的作用特点;汇总了利用基因组编辑技术提高猪生产性能,尤其是改善猪肉品质和抵抗病毒感染的研究进展;分析了目前利用基因组编辑技术推进猪遗传改良所面临的挑战;最后,展望了基于基因组编辑技术的猪遗传改良和品种培育的发展趋势。     

Establishment of an efficient seed fluorescence reporter‐assisted CRISPR/Cas9 gene editing in maize

Genome editing by clustered regularly interspaced short palindromic sequences (CRISPR)/CRISPR‐associated protein 9 (Cas9) has revolutionized functional gene analysis and genetic improvement. While reporter‐assisted CRISPR/Cas systems can greatly facilitate the selection of genome‐edited plants produced via stable transformation, this approach has not been well established in seed crops. Here, we established the seed fluorescence reporter (SFR)‐assisted CRISPR/Cas9 systems in maize (Zea mays L.), using the red fluorescent DsRED protein expressed in the endosperm (En‐SFR/Cas9), embryos (Em‐SFR/Cas9), or both tissues (Em/En‐SFR/Cas9). All three SFRs showed distinct fluorescent patterns in the seed endosperm and embryo that allowed the selection of seeds carrying the transgene of having segregated the transgene out. We describe several case studies of the implementation of En‐SFR/Cas9, Em‐SFR/Cas9, and Em/En‐ SFR/Cas9 to identify plants not harboring the genome‐editing cassette but carrying the desired mutations at target genes in single genes or in small‐scale mutant libraries, and report on the successful generation of single‐target mutants and/or mutant libraries with En‐SFR/Cas9, Em‐SFR/Cas9, and Em/En‐SFR/Cas9. SFR‐assisted genome editing may have particular value for application scenarios with a low transformation frequency and may be extended to other important monocot seed crops.     

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.