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| 1 | 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 |
| 2 | 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 |
| 3 | 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 |
| 4 | Discriminated sgRNAs-Based SurroGate System Greatly Enhances the Screening Efficiency of Plant Base-Edited Cells显示文摘The development of CRISPR/Cas9-mediated base editing has made genomic modification more efficient. However, selection of genetically modified cells from millions of treated cells, especially plant cells, is still challenging. In this study, an efficient surrogate reporter system based on a defective hygromycin resistance gene was established in rice to enrich base-edited cells. After step-by-step optimization, the Discriminated sgRNAs-based SurroGate system (DisSUGs) was established by artificially differentiating the editing abilities of a wild-type single guide RNA (sgRNA) targeting the surrogate reporter gene and an enhanced sgRNA targeting endogenous sites. The DisSUGs enhanced the efficiency of screening base-edited cells by 3- to 5-fold for a PmCDA1-based cytosine-to-tyrosine base editor (PCBE), and 2.5- to 6.5-fold for an adenine base editor (ABE) at endogenous targets. These targets showed editing efficiencies of <25% in the conventional systems. The DisSUGs greatly enhanced the frequency of homozygous substitutions and expanded the activity window slightly for both a PCBE and an ABE. Analyses of the total number of single-nucleotide variants from whole-genome sequencing revealed that, compared with the no-enrichment PCBE strategy, the DisSUGs did not alter the frequency of genome-wide sgRNA-independent off-target mutations, but slightly increased the frequency of target-dependent off-target mutations. Collectively, the DisSUGs developed in this study greatly enhances the efficiency of screening plant base-edited cells and will be a useful system in future applications. | Wen Xu Yongxing Yang Ya Liu Guiting Kang Feipeng Wang Lu Li Xinxin Lv Si Zhao Shuang Yuan Jinling Song Ying Wu Feng Feng Xiaoqing He Chengwei Zhang Wei Song Jiuran Zhao Jinxiao Yang | 2020 | Molecular Plant2020，13，1： | 8 |
| 5 | High-efficiency and multiplex adenine base editing in plants using new TadA variants显示文摘Recently reported adenine base editors(ABEs)exhibit powerful potential for targeted gene correction as well as developing gain-of-function mutants and novel germplasms for both gene function studies and crop breeding.However,editing efficiency varies significantly among different target sites.Here,we investigated the activities of three evolved E.coli adenosine deaminase TadA variants(TadA8e,TadA8.17,and TadA8.20)side-by-side in transgenic rice.We found that TadA8e outperforms TadA8.17 and TadA8.20,and induces efficient A-to-G conversion at all tested sites in the rice genome,including those that were un-editable by ABE7.10 in our previous experiments.Furthermore,V82S/Q154R mutations were incorporated into TadA8e,resulting in a new variant that we named TadA9.Our data show that TadA9 is broadly compatible with CRISPR/SpCas9,CRISPR/SpCas9-NG,and CRISPR/SpRY,as well as CRISPR/ScCas9 nickase systems,achieving comparable or enhanced editing in a larger editing window at diverse PAM sites as compared with TadA8e.Finally,TadA9 was used to simultaneously install novel SNPs in four endogenous herbicide target genes in the commercial rice cultivar Nangeng 46 for potential field application in.weed control.Collectively,we successfully generated a series of novel ABEs that can efficiently edit adenosines in the rice genome.Our findings suggest that TadA9 and TadA8e have great potentials in the development of plant base editors and crop molecular breeding. | Daqi Yan Bin Ren Lang Liu Fang Yan Shaofang Li Guirong Wang Wenxian Sun Xueping Zhou Huanbin Zhou | 2021 | Molecular Plant2021，14，5： | 8 |
| 6 | 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 |
| 7 | Genome editing mediated by SpCas9 variants with broad non-canonical PAM compatibility in plants显示文摘Streptococcus pyogenes Cas9(SpCas9)is the most widely used genome editing tool in plants.The editing induced by SpCas9 strictly requires a canonical NGG protospacer-adjacent motif(PAM),significantly limiting its scope of application.Recently,five SpCas9 variants,SpCas9-NRRH,SpCas9-NRCH,SpCas9-NRTH,SpG,and SPRY,were developed to recognize non-canonical PAMs in human cells.In this study,these variants were engineered for plant genome editing,and their targeted mutagenesis capabilities were comprehensively examined at various canonical and non-canonical PAM sites in rice(Oryza sativa)by stable transformation.Moreover,both cytosine base editors using a rat APOBEC1 or a human APO-BEC3a and adenine base editors using a directly evolved highly compatible TadA*-8e deaminase were developed from these SpCas9 variants.Our results demonstrated that the developed SpCas9 variantsbased base editors readily generated conversions between C.G and T.A in the target sites with noncanonical PAMs in transgenic rice lines.Collectively,the toolbox developed in this study substantially expands the scope of SpCas9-mediated genome editing and will greatly facilitate gene disruption and precise editing in plants. | Juan Li Rongfang Xu Ruiying Qin Xiaoshuang Liu Fanna Kong Pengcheng Wei | 2021 | Molecular Plant2021，14，2： | 6 |
| 8 | Expanding base editing scope to near-PAMless with engineered CRISPR/Cas9 variants in plants显示文摘Dear Editor,Base editors(BEs),including cytosine base editor(CBE)and adenine base editor(ABE),have been widely used to generate irreversible nucleotide substitution in plants and animals.However,their wide applications are largely hindered by the strict NG protospacer adjacent motif(PAM)sequences recognized by Streptococcus pyogenes Cas9(SpCas9)and its engineered variants,such as SpCas9-NG and xCas9(Hua et al.,2019;Ren et al.,2019;Wu et al.,2019;Zhong et al.,2019;Zhang et al.,2020).Most recently,it was reported that three new SpCas9 variants,SpCas9-NRRH,SpCas9-NRTH,and SpCas9-NRCH,could recognize non-G PAMs(NRNH,where R is A or G and H is A,C,or T)in human cells(Miller et al.,2020).Meanwhile,SPRY,another new SpCas9 variant,was developed to greatly expand the editing scope of BEs to nearly PAMless(Walton et al.,2020).In this study,we generated a series of efficient BE toolkits and almost achieved C-to-T mutation without PAM restriction except for NTG PAM,and largely expanded A-to-G mutation scope in stable transformed rice,providing a reference for application in other plants. | Chengwei Zhang Yao Wang Feipeng Wang Si Zhao Jinling Song Feng Feng Jiuran Zhao Jinxiao Yang | 2021 | Molecular Plant2021，14，2： | 4 |
| 9 | 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 |
| 10 | 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 |
| 11 | 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 |
| 12 | CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement显示文摘Since it was first recognized in bacteria and archaea as a mechanism for innate viral immunity in the early 2010 s,clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein(Cas)has rapidly been developed into a robust,multifunctional genome editing tool with many uses.Following the discovery of the initial CRISPR/Cas-based system,the technology has been advanced to facilitate a multitude of different functions.These include development as a base editor,prime editor,epigenetic editor,and CRISPR interference(CRISPRi)and CRISPR activator(CRISPRa)gene regulators.It can also be used for chromatin and RNA targeting and imaging.Its applications have proved revolutionary across numerous biological fields,especially in biomedical and agricultural improvement.As a diagnostic tool,CRISPR has been developed to aid the detection and screening of both human and plant diseases,and has even been applied during the current coronavirus disease 2019(COVID-19)pandemic.CRISPR/Cas is also being trialed as a new form of gene therapy for treating various human diseases,including cancers,and has aided drug development.In terms of agricultural breeding,precise targeting of biological pathways via CRISPR/Cas has been key to regulating molecular biosynthesis and allowing modification of proteins,starch,oil,and other functional components for crop improvement.Adding to this,CRISPR/Cas has been shown capable of significantly enhancing both plant tolerance to environmental stresses and overall crop yield via the targeting of various agronomically important gene regulators.Looking to the future,increasing the efficiency and precision of CRISPR/Cas delivery systems and limiting off-target activity are two major challenges for wider application of the technology.This review provides an in-depth overview of current CRISPR development,including the advantages and disadvantages of the technology,recent applications,and future considerations. | Chao LI Eleanor BRANT Hikmet BUDAK Baohong ZHANG | 2021 | Journal of Zhejiang University-Science B(Biomedicine & Biotechnology)2021，22，4： | 3 |
| 13 | Precision Breeding Made Real with CRISPR: Illustration through Genetic Resistance to Pathogens显示文摘Since its discovery as a bacterial adaptive immune system and its development for genome editing in eukaryotes,the CRISPR technology has revolutionized plant research and precision crop breeding.The CRISPR toolbox holds great promise in the production of crops with genetic disease resistance to increase agriculture resilience and reduce chemical crop protection with a strong impact on the environment and public health.In this review,we provide an extensive overviewon recent breakthroughs in CRISPR technology,including the newly developed prime editing system that allows precision gene editing in plants.We present how each CRISPR tool can be selected for optimal use in accordance with its specific strengths and limitations,and illustrate how the CRISPR toolbox can foster the development of genetically pathogen-resistant crops for sustainable agriculture. | Florian Veillet Mickael Durand Thomas Kroj Stella Cesari Jean-Luc Gallois | 2020 | Plant Communications2020，1，5： | 2 |
| 14 | Single-nucleotide editing for zebra3 and wsl5 phenotypes in rice using CRISPR/Cas9-mediated adenine base editors显示文摘The CRISPR/Cas9-mediated base editing technology can efficiently generate point mutations in the genome without introducing a double-strand break(DSB)or supplying a DNA donor template for homology-directed repair(HDR).In this study,adenine base editors(ABEs)were used for rapid generation of precise point mutations in two distinct genes,OsWSL5,and OsZEBRA3(Z3),in both rice protoplasts and regenerated plants.The precisely engineered point mutations were stably inherited to subsequent generations.These single nucleotide alterations resulted in single amino acid changes and associated wsl5 and z3 phenotypes as evidenced by white stripe leaf and light green/dark green leaf pattern,respectively.Through selfing and genetic segregation,transgene-free,base edited wsl5 and z3 mutants were obtained in a short period of time.We noticed a novel mutation(V540A)in Z3 locus could also mimic the phenotype of Z3 mutation(S542P).Furthermore,we observed unexpected non-A/G or T/C mutations in the ABE editing window in a few of the edited plants.The ABE vectors and the method from this study could be used to simultaneously generate point mutations in multiple target genes in a single transformation and serve as a useful base editing tool for crop improvement as well as basic studies in plant biology. | Kutubuddin A.Molla Justin Shih Yinong Yang | 2020 | aBIOTECH2020，1，2： | 2 |
| 15 | Multiplex and optimization of dCas9-TV-mediated gene activation in plants显示文摘Synthetic gene activators consisting of nucleasedead Cas9(dCas9)for single-guide RNA(sgRNA)-directed promoter binding and a transcriptional activation domain(TAD)represent new tools for gene activation from endogenous genomic locus in basic and applied plant research.However,multiplex gene coactivation by d Cas9-TADs has not been demonstrated in whole plants.There is also room to optimize the performance of these tools.Here,we report that our previously developed gene activator,dCas9-TV,could simultaneously upregulate OsGW7 and OsER1 in rice by up to 3,738 fold,with one sg RNA targeting to each promoter.The gene coactivation could persist to at least the fourth generation.Astonishingly,thepolycistronictRNA-sgRNAexpression under the maize ubiquitin promoter,a Pol II promoter,could cause enormous activation of these genes by up to>40,000-fold in rice.Moreover,the yeast GCN4 coiled coil-mediated dCas9-TV dimerization appeared to be promising for enhancing gene activation.Finally,we successfully introduced a self-amplification loop for dCas9-TV expression in Arabidopsis to promote the transcriptional upregulation of AtFLS2,a previously characterized dCas9-TV-refractory gene with considerable basal expression.Collectively,this work illustrates the robustness of dCas9-TV in multigene coactivation and provides broadly useful strategies for boosting transcriptional activation efficacy of dCas9-TADs in plants. | Xiangyu Xiong Jieping Liang Zhenxiang Li Ben-Qiang Gong Jian-Feng Li | 2021 | Journal of Integrative Plant Biology2021，63，4： | 1 |
| 16 | Expanding plant genome-editing scope by an engineered iSpyMacCas9 system that targets A-rich PAM sequences显示文摘The most popular CRISPR-SpCas9 systemrecognizes canonical NGG protospacer adjacent motifs(PAMs).Previously engineered SpCas9 variants,such as Cas9-NG,favor G-rich PAMs in genome editing.In this manuscript,we describe a new plant genome-editing system based on a hybrid iSpyMacCas9 platform that allows for targeted mutagenesis,C to T base editing,and A to G base editing at A-rich PAMs.This study fills amajor technology gap in the CRISPR-Cas9 system for editing NAAR PAMs in plants,which greatly expands the targeting scope of CRISPR-Cas9.Finally,our vector systems are fully compatible with Gateway cloning and will work with all existing single-guide RNA expression systems,facilitating easy adoption of the systems by others.We anticipate that more tools,such as prime editing,homology-directed repair,CRISPR interference,and CRISPR activation,will be further developed based on our promising iSpyMac-Cas9 platform. | Simon Sretenovic Desuo Yin Adam Levav Jeremy D.Selengut Stephen M.Mount Yiping Qi | 2021 | Plant Communications2021，2，2： | 1 |
| 17 | 基因编辑:将基因生物学用于植物育种的工具显示文摘人口不断增长给世界粮食安全带来了严峻的挑战,植物育种是应对这一挑战的最重要手段之一。过去三十年来,性状形成和调控的新知识(如功能基因组学)和新技术(如生物信息学和表型组学)极大地支持了植物育种的发展。基因编辑,特别是基于CRISPR/Cas技术和其衍生技术,已成为强有力的植物研究技术,可能直接改变植物育种的方法和策略。植物表型性状受编码基因和非编码基因的控制,在本文中,我们提出了编辑这两类基因的不同策略。对于编码基因,其编码蛋白的活性和数量可在转录和转录后水平以及翻译和翻译后水平加以调节,我们由此提出了创造基因功能性变异从而改变性状表型的基因编辑策略。对于非编码基因,则可以采用基因编辑技术对其转录水平或对靶基因的目标序列加以改造,达到产生新的性状的目的。此外,我们还提出了一种基于原生质体的基因编辑方案,使基因编辑技术更适合于植物育种。总之,本文提出了一系列可供植物育种者选择的将基因生物学知识转化为实用育种策略的方案,即基因编辑技术成为将基因生物学知识用于植物育种的技术。 | Yuan-yuan TAN Hao DU Xia WU Yan-hua LIU Meng JIANG Shi-yong SONG Liang WU Qing-yao SHU | 2020 | Journal of Zhejiang University-Science B(Biomedicine & Biotechnology)2020，21，6： | 1 |
| 18 | 基于CRISPR/CasX介导的水稻基因组编辑技术的建立显示文摘Cas蛋白作为核酸酶发挥其切割活性需要识别特定的PAM序列,如SpCas9识别NGG PAM位点,LbCas12a识别TTTV PAM。已挖掘到新的能够识别TTCN PAM序列的蛋白—CasX蛋白,扩展了基因组编辑技术的编辑范围。本研究利用CasX的两个衍生型蛋白PlmCasX和DpbCasX,建立基于CRISPR/CasX介导的水稻基因编辑系统。通过PEG介导的水稻原生质体瞬时表达分析其编辑活性发现,PlmCasX和DpbCasX两个蛋白能够对水稻内源基因OsCPK16实现有效编辑。后通过水稻稳定遗传转化进一步验证,在TTCA PAM识别位点,DpbCasX蛋白对水稻内源基因OsCPK21的编辑效率为17.5%,PlmCasX蛋白对水稻内源基因OsCPK21的编辑效率为66.07%;在TTCG PAM识别位点,PlmCasX蛋白对OsCPK4的编辑效率为23.21%,而DpbCasX蛋白不能实现有效的基因编辑。并且基于MIDAS方法对PlmCasX蛋白的优化并不能提高其编辑活性。本研究证明了CRISPR/CasX系统在水稻中具有编辑活性,且其能识别TTCR PAM这一特性,扩大了基因编辑技术在水稻中的应用范围。 | 李雪琪 张素杰 于曼 黄金光 周焕斌 | 2023 | 生物技术通报2023，39，9： | 0 |
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