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| 1 | CRISPR/Cas系统:RNA靶向的基因组定向编辑新技术显示文摘CRISPR/Cas系统广泛存在于细菌及古生菌中,是机体长期进化形成的RNA指导的降解入侵病毒或噬菌体DNA的适应性免疫系统。对Ⅱ型CRISPR/Cas系统的改造使其成为继锌指核酸酶(ZFNs)和TALE核酸酶(TALENs)以来的另一种对基因组进行高效定点修饰的新技术,与ZFNs和TALENs相比,CRISPR/Cas系统更简单,并且更容易操作。文章重点介绍了Ⅱ型CRISPR/Cas系统的基本结构、作用原理及这一技术在基因组定点修饰中的应用,剖析了该技术可能存在的问题,展望了CRISPR/Cas系统的应用前景,为开展这一领域的研究工作提供参考。 | 李君 张毅 陈坤玲 单奇伟 王延鹏 梁振 高彩霞 | 2013 | 遗传2013，35，11： | 84 |
| 2 | CRISPR/Cas9介导的基因组定点编辑技术显示文摘Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated(Cas)9系统成功被改造为第三代人工核酸内切酶,与锌指核酸内切酶(zinc finger endonuclease,ZFN)和类转录激活因子效应物核酸酶(transcription activator-likeeffector nuclease,TALEN)一样可用于各种复杂基因组的编辑.目前该技术成功应用于人类细胞、斑马鱼和小鼠以及细菌的基因组精确修饰,修饰类型包括基因定点InDel突变、基因定点敲入、两位点同时突变和小片段的缺失.由于其突变效率高、制作简单及成本低的特点,被认为是一种具有广阔应用前景的基因组定点改造分子工具.本文从CRISPR/Cas的研究历史、分类、作用机理以及基因定点修饰应用等方面进行简单介绍,希望能够为在这一领域的科研工作者提供参考. | 方锐 畅飞 孙照霖 李宁 孟庆勇 | 2013 | 生物化学与生物物理进展2013，40，8： | 76 |
| 3 | 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 |
| 4 | Base-Editing-Mediated Artificial Evolution of OsALS1 In Planta to Develop Novel Herbicide-Tolerant Rice Germplasms显示文摘Recently developed CRISPR-mediated base editors,which enable the generation of num erous nucleotide changes in target genomic regions,have been widely adopted for gene correction and generation of crop germ plasms containing im portant gain-of-function genetic variations.How ever,to engineer target genes with unknown functional SNPs remains challenging.To address this issue,we present here abase-e diting-mediated gene evolution(BEMGE)m ethod,employing both Cas9n-based cytosine and adenine base editors as well as a single-guide RNA(sgRNA)library tiling the full-length coding region,for developing novel rice germ plasm swith mutations in any endogenous gene.To this end,OsALS1 was artificially evolved in rice cells using BEMGE through both Agrobacterium-mediated and particle-bom bardment-mediated transform ation.Four different types of amino acid substitutions in the evolved OsALS1,derived from two sites that have never been targeted by natural or human selection during rice dom estication,were identified,conferring varying levels of tolerance to the herbicide bispyribac-sodium.Furtherm ore,the P171F substitution identified in a strong OsALS1 allele was quickly introduced into the commercial rice cultivar Nangeng 46 through precise base editing w ith the corresponding base editor and sgRNA.Collectively,these data indicate great potential of BEMGE in creating important genetic variants of target genes for crop improvement. | Yongjie Kuang Shaofang Li Bin Ren Fang Yan Carl Spetz Xiangju Li Xueping Zhou Huanbin Zhou | 2020 | Molecular Plant2020，13，4： | 37 |
| 5 | CRISPR-Cas系统与细菌和噬菌体的共进化显示文摘细菌在适应噬菌体攻击的过程中,进化了多种防御系统,噬菌体在细菌的选择压力下,也在不断进化反防御策略,双方的这种进化关系与发生机制一直尚不完全清楚。近年在细菌和古细菌中发现一种新的免疫防御系统,即CRISPR-Cas(clustered regularly interspaced short palindromic repeats-CRISPR-associated system)系统。在对其功能和作用机制深入研究的同时,也不断地揭示了细菌和噬菌体之间的共进化关系。为此,文章在介绍原核细胞中CRISPR-Cas系统介导的免疫机制基础上,重点综述了CRISPR系统在细菌和噬菌体进化中的作用。 | 李铁民 杜波 | 2011 | 遗传2011，33，3： | 33 |
| 6 | 沙门氏菌分型研究进展显示文摘沙门氏菌是重要的致病菌之一,该菌属型别繁多。沙门氏菌的分型方法可分为以表型特征为依据的表型分型方法和以基因特征为依据的分子分型方法。沙门氏菌的表型分型主要有血清分型和噬菌体分型。分子分型主要有分子血清分型、脉冲场凝胶电泳(Pulsed field gel electrophoresis,PFGE)、多位点序列分析(Multi-locus sequence typing,MLST)、多位点可变重复序列分析(Multi-locus variable numbers tandem repeat analysis,MLVA)以及成簇的规律间隔的短回文重复序列分析(Clustered regularly interspaced short palindromic repeats,CRISPR)等。与表型分型相比,分子分型技术具有快速、准确、重复性好等特点,现已得到广泛应用。其中,分子血清分型是鉴定沙门氏菌血清型的新方法,PFGE被认为是病原微生物分子分型的'金标准',MLST和MLVA以其分辨率高、可重复性好和可比性强等优势满足了全球流行病学发展的要求,能实现序列数据交换和共享,成为新一代的分子分型新工具,而近年发现的CRISPR分型对同源性较高的同种血清型具有较高的分型能力。但每种分型方法也有各自的优缺点和使用条件及适用范围,因此可以根据菌株特性、分型目的和实验室拥有的条件选择最合适的分型方法。 | 陈玲 张菊梅 杨小鹃 吴清平 | 2016 | 微生物学通报2016，43，3： | 29 |
| 7 | CRISPR/Cas系统在植物基因组编辑中的应用显示文摘在基因组水平上进行植物基因人工改造对农作物的改良具有重要意义。一直以来,科学家都未发现有效的植物基因组编辑的方法。最近发现的CRISPR/Cas系统因其简易和有效性被广泛应用于包括植物在内的多种生物的基因组编辑中。CRISPR/Cas系统仅需要短RNA和核酸酶就可以对特定的靶标基因进行突变。目前CRISPR/Cas系统已成功在拟南芥(Arabidopsis thaliana)、烟草(Nicotiana benthamiana)、甜橙(Citrus sinensis)、水稻(Oryza sativa)、小麦(Triticum aestivum)、高粱(Sorghum bicolor)、玉米(Zea mays)以及苔藓植物地钱(Marchantia polymorpha)等植物中实现了定点基因组编辑。综述将对CRISPR/Cas系统在植物中的最新研究进行全面的总结和讨论。 | 瞿礼嘉 郭冬姝 张金喆 秦跟基 | 2015 | 生命科学2015，27，1： | 26 |
| 8 | 新一代基因组编辑系统CRISPR/Cpf1显示文摘CRISPR/Cas系统几乎存在于所有的细菌和古菌中,是用来抵御外来病毒和噬菌体入侵的获得性免疫防御机制。2012年起CRISPR/Cas9被改造为基因编辑工具,并衍生出一系列高效、便捷的基因编辑工具,迅速在基础理论、基因诊断和临床治疗等研究领域中得到广泛应用。然而,CRISPR/Cas9也存在细胞毒性、脱靶效应和基因插入困难等一些亟待解决的问题,在一定程度上限制了CRISPR/Cas9的应用。Cpf1是2015年报道的一种新型CRISPR效应蛋白,具有许多与Cas9不同的特性,有利于克服CRISPR/Cas9应用中的一些限制。本文综述了近两年来对CRISPR/Cpf1的研究进展和应用,并对其应用前景和发展方向进行了展望。 | 杨帆 李寅 | 2017 | 生物工程学报2017，33，3： | 20 |
| 9 | 利用CRISPR/Cas9敲除大鼠胰岛素受体底物1(Irs1)基因显示文摘目的为研究胰岛素受体底物1(Irs1)基因与代谢病之间的关系,我们利用CRISPR/Cas9系统敲除大鼠Irs1基因,为研究代谢病提供基因敲除大鼠。方法针对Irs1第一外显子,设计CRISPR/Cas9作用靶点,构建sgRNA表达质粒。利用T7 RNA聚合酶体外转录sgRNA和Cas9。将Cas9 mRNA和sgRNA混合物注射入SD大鼠的受精卵中,实现靶基因敲除。用T7EN1实验初步检测靶基因的修饰情况,再经过测序分析确定突变。结果获得了5个在Irs1基因突变的首建鼠,突变效率为83%。结论得到了稳定遗传的Irs1基因敲除大鼠。 | 马元武 马婧 路迎冬 陈炜 张旭 于磊 张连峰 | 2014 | 中国比较医学杂志2014，24，3： | 18 |
| 10 | Cas9-NG Greatly Expands the Targeting Scope of the Genome-Editing Toolkit by Recognizing NG and Other Atypical PAMs in Rice显示文摘CRISPR technologies enabling precise genome manipulation are valuable for gene function studies and molecular crop breeding. However, the requirement of a protospacer adjacent motif (PAM)y such as NGG and TTN, for Cas protein recognition restricts the selection of targetable genomic loci in practical applications of CRISPR technologies. Recently Cas9-NG, which recognizes a minimal NG PAM, was reported to expand the targeting space of genome editing in human cells, but it remains unclear whether this Cas9 variant can be used in plants. In this study, we evaluated the nuclease activity of Cas9-NG toward various NGN PAMs by targeting endogenous genes in transgenic rice. We found that Cas9-NG edits all NGG, NGA, NGT, and NGC sites with impaired activity, while the gene-edited plants were dominated by monoallelic mutations. Cas9-NG-engineered base editors were then developed and used to generate O s B Z R I gainof- function plants that can not be created by other available Cas9-engineered base editors. Moreover, we showed that a Cas9-NG-based transcriptional activator efficiently upregulated the expression of endogenous target genes in rice. In addition, we discovered that Cas9-NG recognizes NAC, NTG, NTT, and NCG apart from NG PAM. Together, these findings demonstrate that Cas9-NG can greatly expand the targeting scope of genome-editing tools, showing great potential for targeted genome editing, base editing, and genome regulation in plants. | Bin Ren Lang Liu Shaofang Li Yongjie Kuang Jingwen Wang Dawei Zhang Xueping Zhou Honghui Lin Huanbin Zhou | 2019 | Molecular Plant2019，12，7： | 18 |
| 11 | 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 |
| 12 | 志贺菌CRISPR的检测及其与耐药的关系显示文摘【目的】检测志贺菌成簇规律间隔的短回文重复序列(Clustered regularly interspaced short palindromic repeats,CRISPR),并分析其与志贺菌耐药的关系。【方法】根据CRISPR DB数据库公布的志贺菌确定的CRISPR结构序列CRISPR-S2、CRISPR-S4和可能的CRISPR结构序列CRISPR-S1、CRISPR-S3设计四对引物,对60株志贺菌进行PCR扩增。采用CRISPR Finder分析CRISPR,采用改良K-B药敏纸片法检测志贺菌耐药情况,并分析CRISPR-S4与耐药的关系。【结果】确定的CRISPR结构的总阳性率为95%,四个CRISPR位点组成12种CRISPR谱型(A-L),除K型外均含确定的CRISPR结构,新发现1种重复序列和12种间隔序列。60株志贺菌的多重耐药率为53.33%。CRISPR-S4阳性菌株与阴性菌株之间,耐药的分布差异无统计学意义,但多重耐药菌株和耐TE菌株CRISPR-S4的重复序列多为R4.1,其3'末端缺失碱基AC;多重耐药菌株CRISPR-S4的间隔序列多为Sp5.1、Sp6.1和Sp7。【结论】CRISPR在志贺菌中广泛分布。CRISPR重复序列的变异和间隔序列的多样性可能与志贺菌耐药有关。 | 王琳琳 王颖芳 段广才 薛泽润 郭向娇 王鹏飞 郗园林 杨海燕 | 2015 | 微生物学报2015，55，4： | 15 |
| 13 | CRISPR-Cas9基因编辑技术在病毒感染疾病治疗中的应用显示文摘CRISPR-Cas9基因编辑技术是基于细菌或古细菌CRISPR介导的获得性免疫系统衍生而来,由一段RNA通过碱基互补配对识别DNA,指导Cas9核酸酶切割识别的双链DNA,诱发同源重组或非同源末端链接,进而实现在目的 DNA上进行编辑。病毒通过特异的受体侵染细胞,其基因组在细胞内发生复制、转录、翻译等过程完成其生活周期,某些DNA病毒或逆转录病毒基因组会整合到宿主基因组中。基因治疗是病毒感染疾病治疗的新趋势。因此,基因编辑技术在持续感染的病毒或潜伏感染病毒疾病治疗中具有重大的潜在意义。文章主要从CRISPR-Cas9作用机制以及在病毒感染疾病治疗中的应用等方面进行了综述。 | 殷利眷 胡斯奇 郭斐 | 2015 | 遗传2015，37，5： | 15 |
| 14 | Characterization and Evaluation of OsLCT1 and OsN ramp5 Mutants Generated Through CRISPR/Cas9-Mediated Mutagenesis for Breeding Low Cd Rice显示文摘To explore how rice(Oryza sativa L.) can be safely produced in Cd-polluted soil, OsLCT1 and OsNramp5 mutant lines were generated by CRISPR/Cas9-mediated mutagenesis. One of OsLCT1 mutant(lct1×1) and two of OsNramp5 mutants(nramp5×7 and nramp5×9) were evaluated for grain Cd accumulation and agronomic performances. In paddy field soil containing approximately 0.9 mg/kg Cd, lct1×1 grains contained approximately 40%(0.17 mg/kg) of the Cd concentration of the wild type parental line, less than the China National Food Safety Standard(0.20 mg/kg). Both OsNramp5 mutants showed low grain Cd accumulation(< 0.06 mg/kg) in the paddy(approximately 0.9 mg/kg Cd) or in pots in soil spiked with 2 mg/kg Cd. However, only nramp5×7 showed normal growth and yield, whereas the growth of nramp5×9 was severely impaired. The study showed that lct1×1 could be used to produce rice grains safe for human consumption in lightly contaminated paddy soils and nramp5×7 used in soils contaminated by much higher levels of Cd. | LIU Songmei JIANG Jie LIU Yang MENG Jun XU Shouling TAN Yuanyuan LI Youfa SHU Qingyao HUANG Jianzhong | 2019 | Rice science2019，26，2： | 15 |
| 15 | 基因编辑技术及其在中国的研究发展显示文摘基因编辑技术是一种能够对生物体的基因组及其转录产物进行定点修饰或者修改的技术,早期基因编辑技术包括归巢内切酶、锌指核酸内切酶和类转录激活因子效应物。近年来,以CRISPR/Cas9系统为代表的新型技术使基因编辑的研究和应用领域得以迅速拓展。本文对基因编辑技术的原理、技术发展及其应用进行了阐述,对我国在基因编辑机制研究及技术发展、基因编辑动植物模型构建、基因治疗等领域的研究进展进行了回顾,并对基因技术的发展前景及趋势进行了展望。 | 陈一欧 宝颖 马华峥 伊宗裔 周卓 魏文胜 | 2018 | 遗传2018，40，10： | 14 |
| 16 | 基因编辑技术伦理治理探讨显示文摘基因编辑技术已成为生命科学领域重要的颠覆性技术之一。随着该技术在生物医学研究、医疗健康、农业和食品等领域的应用,相关伦理问题也备受关注。文章概述了近年来涉及伦理问题的基因编辑技术研究和应用进展,重点梳理国际上相关问题的讨论及在伦理治理方面的态度和探索;最后,结合我国对于基因编辑技术人体应用的治理现状,以及近年来的相关讨论和举措,初步提出我国基因编辑技术伦理治理体系建设的建议,以供决策参考。 | 王慧媛 李鹏飞 徐丽娟 张丽雯 贺彩红 范月蕾 于建荣 许智宏 | 2021 | 中国科学院院刊2021，36，11： | 13 |
| 17 | CRISPR technology is revolutionizing the improvement of tomato and other fruit crops显示文摘Fruits are major sources of essential nutrients and serve as staple foods in some areas of the world.The increasing human population and changes in climate experienced worldwide make it urgent to the production of fruit crops with high yield and enhanced adaptation to the environment,for which conventional breeding is unlikely to meet the demand.Fortunately,clustered regularly interspaced short palindromic repeat(CRISPR)technology paves the way toward a new horizon for fruit crop improvement and consequently revolutionizes plant breeding.In this review,the mechanism and optimization of the CRISPR system and its application to fruit crops,including resistance to biotic and abiotic stresses,fruit quality improvement,and domestication are highlighted.Controversies and future perspectives are discussed as well. | Tian Wang Hongyan Zhang Hongliang Zhu | 2019 | Horticulture Research2019，6，1： | 13 |
| 18 | Increasing yield potential through manipulating of an ARE1 ortholog related to nitrogen use efficiency in wheat by CRISPR/Cas9显示文摘Wheat(Triticum aestivum L.)is a staple food crop consumed by more than 30%of world population.Nitrogen(N)fertilizer has been applied broadly in agriculture practice to improve wheat yield to meet the growing demands for food production.However,undue N fertilizer application and the low N use efficiency(NUE)of modern wheat varieties are aggravating environmental pollution and ecological deterioration.Under nitrogen-limiting conditions,the rice(Oryza sativa)abnormal cytokinin response1 repressor1(are1)mutant exhibits increased NUE,delayed senescence and consequently,increased grain yield.However,the function of ARE1 ortholog in wheat remains unknown.Here,we isolated and characterized three TaARE1 homoeologs from the elite Chinese winter wheat cultivar ZhengMai 7698.We then used CRISPR/Cas9-mediated targeted mutagenesis to generate a series of transgene-free mutant lines either with partial or triple-null taare1 alleles.All transgene-free mutant lines showed enhanced tolerance to N starvation,and showed delayed senescence and increased grain yield in field conditions.In particular,the AABBdd and aabbDD mutant lines exhibited delayed senescence and significantly increased grain yield without growth defects compared to the wild-type control.Together,our results underscore the potential to manipulate ARE1 orthologs through gene editing for breeding of high-yield wheat as well as other cereal crops with improved NUE. | Jiahui Zhang Huating Zhang Shaoya Li Jingying Li Lei Yan Lanqin Xia | 2021 | Journal of Integrative Plant Biology2021，63，9： | 13 |
| 19 | CRISPR基因编辑技术在微生物合成生物学领域的研究进展显示文摘微生物合成生物学是一门新兴的交叉学科,其主要目的是通过改造或创制微生物细胞,使微生物具有特定的生理功能或生产目标产物,因此需要高效、快速、精准的基因操作工具。CRISPR技术是一种成本低、操作简便、效率高、功能多样的基因编辑技术,近年来被广泛应用于合成生物学、代谢工程和医学研究等领域,极大地促进了这些领域的发展。本文简述了CRISPR基因编辑技术的发展历史及其作用机制,重点介绍了近年来CRISPR/Cas9技术在微生物合成生物学领域研究和应用的进展,列举了CRISPR/Cas9技术在微生物合成生物学中生产目标产品的研究,总结了由CRISPR/Cas9技术衍生出的CRISPR/Cas12a、CRISPR/Cas13等技术在微生物合成生物学领域的研究及应用,提出了CRISPR基因编辑技术现存的PAM依赖性、脱靶效应、安全性和应用广泛性等问题,最后展望了该技术在构建高效微生物细胞工厂生产高附加值化合物的发展前景和创造更多适合生产高附加值产品的底盘生物的研究方向。 | 李洋 申晓林 孙新晓 袁其朋 闫亚军 王佳 | 2021 | 合成生物学2021，2，1： | 11 |
| 20 | Targeted mutagenesis of amino acid transporter genes for rice quality improvement using the CRISPR/Cas9 system显示文摘High grain protein content(GPC) reduces rice eating and cooking quality(ECQ). We generated OsAAP6 and OsAAP10 knockout mutants in three high-yielding japonica varieties and one japonica line using the CRISPR/Cas9 system. Mutation efficiency varied with genetic background in the T_0 generation, and GPC in the T_1 generation decreased significantly,owing mainly to a reduction in glutelin content. Amylose content was down-regulated significantly in some Osaap6 and all Osaap10 mutants. The increased taste value of these mutants was supported by Rapid Visco Analysis(RVA) profiles, which showed higher peak viscosity and breakdown viscosity and lower setback viscosity than the wild type. There were no significant deficiencies in agronomic traits of the mutants. Targeted mutagenesis of OsAAP6 and OsAAP10, especially OsAAP10, using the CRISPR/Cas9 system can rapidly reduce GPC and improve ECQ of rice, providing a new strategy for the breeding cultivars with desired ECQ. | Shiyu Wang Yihao Yang Min Guo Chongyuan Zhong Changjie Yan Shengyuan Sun | 2020 | The Crop Journal2020，8，3： | 11 |
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