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| 1 | CRISPR/Cas植物基因组编辑技术研究进展显示文摘基因编辑技术的发展与应用为植物功能基因研究和作物遗传改良提供了重要的技术支撑。近年诞生的CRISPR/Cas 基因编辑系统(主要包括 CRISPR/Cas9 和 CRISPR/Cas12a)与其他的基因编辑技术相比,具有操作简单、效率高等优势,因此在动植物中均得到广泛应用。本文结合 CRISPR/Cas 基因编辑技术体系的发展历史及最新研究进展,着重介绍了该技术在植物领域中的应用范围和发展方向,以及基因编辑植物的靶点分析方法;对目前CRISPR/Cas 基因编辑技术体系存在的问题进行了分析并提出了改进策略。 | 刘耀光 李构思 张雅玲 陈乐天 | 2019 | 华南农业大学学报2019，40，5： | 56 |
| 2 | Hi-TOM: a platform for high-throughput tracking of mutations induced by CRISPR/Cas systems显示文摘The CRISPR/Cas system has been extensively applied to make precise genetic modifications in various organisms. Despite its importance and widespread use, large-scale mutation screening remains time-consuming, labour-intensive and costly. Here, we developed Hi-TOM(available at http://www.hi-tom.net/hi-tom/), an online tool to track the mutations with precise percentage for multiple samples and multiple target sites. We also described a corresponding next-generation sequencing(NGS) library construction strategy by fixing the bridge sequences and barcoding primers. Analysis of the samples from rice, hexaploid wheat and human cells reveals that the Hi-TOM tool has high reliability and sensitivity in tracking various mutations, especially complex chimeric mutations frequently induced by genome editing. Hi-TOM does not require special design of barcode primers,cumbersome parameter configuration or additional data analysis. Thus, the streamlined NGS library construction and comprehensive result output make Hi-TOM particularly suitable for high-throughput identification of all types of mutations induced by CRISPR/Cas systems. | Qing Liu Chun Wang Xiaozhen Jiao Huawei Zhang Lili Song Yanxin Li Caixia Gao Kejian Wang | 2019 | Science China(Life Sciences)2019，62，1： | 37 |
| 3 | 碱基编辑系统研究进展显示文摘碱基编辑技术(base editing)是基于CRISPR/Cas系统发展起来的新型靶基因修饰技术,目前依据碱基修饰酶的不同可分为胞嘧啶碱基编辑器(cytosine base editor,CBE)和腺嘌呤碱基编辑器(adenine base editor,ABE)。这两类碱基编辑系统利用胞嘧啶脱氨酶或人工进化的腺嘌呤脱氨酶对靶位点进行精准的碱基编辑,最终可以分别实现C-T(G-A)或A-G(T-C)的碱基替换。碱基编辑技术自2016年被开发以来,因其高效、不依赖DNA双链断裂产生、无需供体DNA参与等优势,已经成功应用在各种动物、植物及其他生物中,为基因治疗及精准作物育种等领域提供了重要技术支撑。本文从碱基编辑技术的特点、开发过程、优化、应用、脱靶效应及改善策略等方面进行了系统介绍,最后对未来需要迫切解决的一些问题进行了分析和展望,以期为相关领域的科研人员进一步了解、使用及优化碱基编辑系统提供参考。 | 宗媛 高彩霞 | 2019 | 遗传2019，41，9： | 37 |
| 4 | Manipulating mRNA splicing by base editing in plants显示文摘Precursor-mRNAs(pre-mRNA) can be processed into one or more mature m RNA isoforms through constitutive or alternative splicing pathways. Constitutive splicing of pre-mRNA plays critical roles in gene expressional regulation, such as intronmediated enhancement(IME), whereas alternative splicing(AS) dramatically increases the protein diversity and gene functional regulation. However, the unavailability of mutants for individual spliced isoforms in plants has been a major limitation in studying the function of mRNA splicing. Here, we describe an efficient tool for manipulating the splicing of plant genes. Using a Cas9-directed base editor, we converted the 5′ splice sites in four Arabidopsis genes from the activated GT form to the inactive AT form. Silencing the AS of HAB 1.1(encoding a type 2 C phosphatase) validated its function in abscisic acid signaling, while perturbing the AS of RS31 A revealed its functional involvement in plant response to genotoxic treatment for the first time. Lastly,altering the constitutive splicing of Act2 via base editing facilitated the analysis of IME. This strategy provides an efficient tool for investigating the function and regulation of gene splicing in plants and other eukaryotes. | Chenxiao Xue Huawei Zhang Qiupeng Lin Rong Fan Caixia Gao | 2018 | Science China(Life Sciences)2018，61，11： | 18 |
| 5 | Gene editing in plants:progress and challenges显示文摘The clustered regularly interspaced short palindromic repeat(CRISPR)-associated protein 9(Cas9)genome editing system is a powerful tool for targeted gene modifications in a wide range of species,including plants.Over the last few years,this system has revolutionized the way scientists perform genetic studies and crop breeding,due to its simplicity,flexibility,consistency and high efficiency.Considerable progress has been made in optimizing CRISPR/Cas9 systems in plants,particularly for targeted gene mutagenesis.However,there are still a number of important challenges ahead,including methods for the efficient delivery of CRISPR and other editing tools to most plants,and more effective strategies for sequence knock-ins and replacements.We provide our viewpoint on the goals,potential concerns and future challenges for the development and application of plant genome editing tools. | Yanfei Mao Jose Ramon Botella Yaoguang Liu Jian-Kang Zhu | 2019 | National Science Review2019，6，3： | 17 |
| 6 | ALS抑制剂类除草剂的抗性研究概述显示文摘乙酰乳酸合成酶(ALS)是支链氨基酸生物合成途径中的关键酶之一,是磺酰脲类除草剂的作用靶标。针对目前市场上使用的4种ALS除草剂,对ALS抑制剂类除草剂的抗性杂草问题, ALS氨基酸序列中关键位点替换对ALS酶活性的影响与ALS基因编辑等方面的相关研究进展进行了概述。 | 辛洁 徐小博 王磊 朱晔荣 王勇 | 2019 | 安徽农业科学2019，47，4： | 11 |
| 7 | CRISPR-Cas系统在植物基因组编辑中的研究进展显示文摘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。 | 王春 王克剑 | 2017 | 生物工程学报2017，33，10： | 10 |
| 8 | Development of Plant Prime-Editing Systems for Precise Genome Editing显示文摘Prime-editing systems have the capability to perform efficient and precise genome editing in human cells.In this study,we first developed a plant prime editor 2(pPE2)system and test its activity by generating a targeted mutation on an HPT^(-ATG) reporter in rice.Our results showed that the pPE2 system could induce programmable editing at different genome sites.In transgenic T0 plants,pPE2-generated mutants occurred with 0%–31.3%frequency,suggesting that the efficiency of pPE2 varied greatly at different genomic sites and with prime-editing guide RNAs of diverse structures.To optimize editing efficiency,guide RNAs were introduced into the pPE2 system following the PE3 and PE3b strategy in human cells.However,at the genomic sites tested in this study,pPE3 systems generated only comparable or even lower editing frequencies.Furthemore,we developed a surrogate pPE2 system by incorporating the HPT^(-ATG) reporter to enrich the prime-edited cells.The nucleotide editing was easily detected in the resistant calli transformed with the surrogate pPE2 system,presumably due to the enhanced screening efficiency of edited cells.Taken together,our results indicate that plant prime-editing systems we developed could provide versatile and flexible editing in rice genome. | Rongfang Xu Juan Li Xiaoshuang Liu Tiaofeng Shan Ruiying Qin Pengcheng Wei | 2020 | Plant Communications2020，1，3： | 10 |
| 9 | Base editing in plants: Current status and challenges显示文摘Genome editing technologies have revolutionized the field of plant science by enabling targeted modification of plant genomes and are emerging as powerful tools for both plant gene functional analyses and crop improvement. Although homology-directed repair(HDR)is a feasible approach to achieve precise gene replacement and base substitution in some plant species, the dominance of the non-homologous end joining pathway and low efficiency of HDR in plant cells have limited its application. Base editing has emerged as an alternative tool to HDR-mediated replacement, facilitating precise editing of plant genome by converting one single base to another in a programmable manner without a doublestranded break and a donor repair template. In this review, we summarize the latest developments in base-editing technologies as well as their underlying mechanisms. We review current applications of these technologies in plant species. Finally, we address the challenges and future perspectives of this emerging technology in plants. | Sutar Suhas Bharat Shaoya Li Jingying Li Lei Yan Lanqin Xia | 2020 | The Crop Journal2020，8，3： | 8 |
| 10 | 植物基因组编辑检测方法显示文摘以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。 | 刘春霞 耿立召 许建平 | 2018 | 遗传2018，40，12： | 5 |
| 11 | Genome editing for plant research and crop improvement显示文摘The advent of clustered regularly interspaced short palindromic repeat(CRISPR) has had a profound impact on plant biology, and crop improvement. In this review, we summarize the state-of-the-art development of CRISPR technologies and their applications in plants, from the initial introduction of random small indel(insertion or deletion) mutations at target genomic loci to precision editing such as base editing, prime editing and gene targeting. We describe advances in the use of class 2, types II, V, and VI systems for gene disruption as well as for precise sequence alterations, gene transcription, and epigenome control. | Xiangqiang Zhan Yuming Lu Jian-Kang Zhu Jose Ramon Botella | 2021 | Journal of Integrative Plant Biology2021，63，1： | 5 |
| 12 | CRISPR/Cas9基因组编辑技术及其在作物遗传改良中的应用进展显示文摘基因组编辑技术可对作物靶标基因进行定点删除、替换、插入等遗传操作,现已被广泛应用于作物基因功能解析和品种改良。简要介绍了基因编辑技术的发展历程,阐述了基因编辑技术CRISPR/Cas9系统的工作原理,以及基于CRISPR/Cas9的单碱基编辑、引导编辑器等一系列衍生编辑工具,同时总结介绍了近年来上述基因组技术在不同农作物性状改良中的应用实例。最后探讨了CRISPR/Cas9技术目前存在的一些局限性及其解决方法,并且对基因编辑技术未来在作物中的应用进行了展望。 | 王莹婕 马玲玲 梁振 | 2021 | 山西农业科学2021，49，12： | 5 |
| 13 | Generating broad-spectrum tolerance to ALS-inhibiting herbicides in rice by base editing显示文摘Herbicide-tolerant rice varieties generated by genome editing are highly desirable for weed control.We have used a cytosine base editor to create a series of missense mutations in the P171 and/or G628 codons of the acetolactate synthase(ALS)gene to confer herbicide tolerance in rice.The four different missense mutations in the P171 codon,P171S,P171A,P171Yand P171F,exhibited different patterns of tolerance towards five representative herbicides from five chemical families of ALS inhibitors.For example,P171S and P171A had lower levels of tolerance than P171Y and P171F to bispyribac but not to the other herbicides.Interestingly,a novel triple mutant(P171F/G628E/G629S)had the highest tolerance to all five tested herbicides.Field trials showed that both P171F and P171F/G628E/G629S could potentially be used with nicosulfuron.Our work illustrates an effective way of using base editing to generate herbicide tolerance in elite rice varieties. | Rui Zhang Sha Chen Xiangbing Meng Zhuangzhuang Chai Delin Wang Yuge Yuan Kunling Chen Linjian Jiang Jiayang Li Caixia Gao | 2021 | Science China(Life Sciences)2021，64，10： | 4 |
| 14 | CRISPR-Cas系统在植物中的研究进展与监管政策显示文摘基因编辑技术作为一种颠覆性新技术,现已广泛应用于作物的遗传改良,显示出巨大的发展潜力和应用价值。各国在加快技术研发的同时也十分关注其可能带来的安全性问题,相继出台了基因编辑作物的安全监管政策。综述了目前常用的CRISPR基因编辑系统的原理,最新开发的一系列CRISPR变体,CRISPR系统在植物中的应用,基因编辑植物检测方法及国际上的相关监管政策,以期为我国基因编辑作物监管政策的制定提供理论数据。 | 刘肖静 王旭静 王志兴 | 2021 | 生物技术进展2021，11，1： | 4 |
| 15 | 碱基编辑技术及其在作物遗传改良中的应用综述显示文摘碱基编辑技术是以CRISPR/Cas系统为基础开发的一种能够对基因组进行定点精准编辑的新技术,包括胞嘧啶碱基编辑系统(cytosine base editor,CBE),腺嘌呤碱基编辑系统(adenine base editor,ABE)以及引导编辑系统(primeediting,PE)。胞嘧啶碱基编辑系统可以将基因组靶位点处的C/G转换为T/A,腺嘌呤碱基编辑系统可以将靶位点处的A/T转变为G/C,而引导编辑系统则可以实现所有12种类型(C-T、G-A、A-G、T-C、C-A、C-G、G-C、G-T、A-C、A-T、T-A、T-G)碱基的任意替换以及碱基的插入和删除。本文中系统介绍了这3种碱基编辑系统的原理、开发过程、各自的优缺点以及在作物遗传改良中的应用和发展,并展望了碱基编辑技术在农作物育种中的应用前景。 | 李国斌 艾国 韦静 张俊红 | 2021 | 园艺学报2021，48，4： | 4 |
| 16 | 乙酰羟酸合酶抑制剂类除草剂的植物抗性机制显示文摘乙酰羟酸合酶(AHAS)抑制剂类除草剂已被广泛用于农业生产,然而使用过程中可能对部分敏感农作物产生药害,因此创制对不同类别除草剂具有抗性的一系列作物新品种至关重要。本文将从AHAS抑制剂类除草剂的类别与特点、AHAS靶酶的特性及其在支链氨基酸合成中的作用、除草剂的靶标抗性与非靶标抗性机制等方面分析国内外研究现状与未来发展动态,以期为农作物除草剂抗性性状的遗传改良和开发应用提供参考。 | 徐倩玉 兰玉 刘嘉欣 周新宇 张刚 郑志富 | 2019 | 作物学报2019，45，9： | 3 |
| 17 | Development and molecular analysis of a novel acetohydroxyacid synthase rapeseed mutant with high resistance to sulfonylurea herbicides显示文摘With the increasing promotion of simplified rapeseed cultivation in recent years,the development of cultivars with high resistance to herbicides is urgently needed.We previously developed M342,which shows sulfonylurea herbicide resistance,by targeting acetohydroxyacid synthase(AHAS),a key enzyme in branched-chain amino acid synthesis.In the present study,we used a progeny line derived from M342 for an additional round of ethyl methane sulfonate mutagenesis,yielding the novel mutant DS3,which harbored two mutations in AHAS genes and showed high sulfonylurea resistance.One mutation was the substitution Trp574 Leu,as in M342,according to Arabidopsis protein sequencing.The other site was a newly recognized substitution,Pro197 Leu.A KASP marker targeting Pro197 Leu was developed and reliably predicted the response to sulfonylurea herbicides in the F2 population.The combination of Trp574 Leu and Pro197 Leu in DS3 produced a synergistic effect that greatly increased herbicide resistance.Analysis of the protein structures of AHAS1 and AHAS3 in wild-type and single-gene mutant plants revealed three-dimensional protein conformational changes that could account for differences in herbicide resistance characteristics including toxicity tolerance,AHAS enzyme activity,and AHAS gene expression. | Yue Guo Changle Liu Weihua Long Jianqin Gao Jiefu Zhang Song Chen Huiming Pu Maolong Hu | 2022 | The Crop Journal2022，10，1： | 3 |
| 18 | The CRISPR/Cas9 revolution continues: From base editing to prime editing in plant science显示文摘The ability to precisely inactivate or modify genes in model organisms helps us understand the mysteries of life. Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9), a revolutionary technology that could generate targeted mutants, has facilitated notable advances in plant science. Genome editing with CRISPR/Cas9 has gained great popularity and enabled several technical breakthroughs. Herein, we briefly introduce the CRISPR/Cas9, with a focus on the latest breakthroughs in precise genome editing(e.g., base editing and prime editing), and we summarize various platforms that developed to increase the editing efficiency, expand the targeting scope, and improve the specificity of base editing in plants. In addition, we emphasize the recent applications of these technologies to plants. Finally, we predict that CRISPR/Cas9 and CRISPR/Cas9-based genome editing will continue to revolutionize plant science and provide technical support for sustainable agricultural development. | Yan Li Wenjing Li Jun Li | 2021 | Journal of Genetics and Genomics2021，48，8： | 3 |
| 19 | Increasing fidelity and efficiency by modifying cytidine base-editing systems in rice显示文摘The efficiency of plant cytidine base-editing systems is limited, and unwanted mutations frequently occur in transgenic plants. We increased the cytidine editing frequency and fidelity of the plant base editor 3(BE3) and targeted activation-induced cytidine deaminase(CDA)(target-AID) systems by coexpressing three copies of free uracil–DNA glycosylase(UDG) inhibitor(UGI). The editing efficiency of the improved BE3 and CDA systems reached as high as 88.9% and 85.7%, respectively, in regenerated rice plants, with a very low frequency of unwanted mutations. The low editing frequency of the BE3 system in the GC context could be overcome by the modified CDA system. These results provide a highfidelity and high-efficiency solution for rice genomic base editing. | Ruiying Qin Shengxiang Liao Juan Li Hao Li Xiaoshuang Liu Jianbo Yang Pengcheng Wei | 2020 | The Crop Journal2020，8，3： | 3 |
| 20 | 植物CRISPR/Cas技术研究进展及其在林业科学研究中的应用显示文摘近年来,CRISPR/Cas系统在基因编辑领域展现出强大的定点编辑能力,在植物基础研究和分子育种等方面发挥了不可替代的作用.本文综述了CRISPR/Cas系统的发展历程,及其在植物研究应用中的遗传转化方法和鉴定方法等;同时还介绍了这种基因编辑技术在植物领域的多种研究新方向;并详细概述了CRISPR/Cas系统在林木基因功能分析鉴定等基础科研方面的应用情况;最后展望了该技术在林业科学研究中的应用前景,以期为后续相关的林木遗传改良等研究提供参考. | 胡凯强 廖家凯 席飞虎 高鹏飞 陈凯 魏文桃 丁家治 苗苗 顾连峰 | 2021 | 福建农林大学学报(自然科学版)2021，50，3： | 2 |
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