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洋山港宏病毒组分析揭示crispr-Cas系统病毒靶标序列的特异性

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生物化学与生物物理进展 2023年第2期50卷 358-370页

作者：李聪田敬孜王永杰上海海洋大学食品学院上海201306 农业农村部水产品质量安全贮藏保鲜风险评估实验室(上海) 上海201306 青岛海洋国家实验室海洋生物学与生物技术功能实验室青岛266200

目的为了探究crispr-Cas系统的靶标特点。方法运用了包括BALST在内的多种生物信息学分析技术和方法对洋山港宏病毒组数据进行了分析。结果洋山港宏病毒组数据集中共有25 391条双链DNA病毒序列,其中238条序列上的265个开放读码框(ORF)... 详细信息

目的为了探究crispr-Cas系统的靶标特点。方法运用了包括BALST在内的多种生物信息学分析技术和方法对洋山港宏病毒组数据进行了分析。结果洋山港宏病毒组数据集中共有25 391条双链DNA病毒序列,其中238条序列上的265个开放读码框(ORF)与134条规律间隔短回文重复(crispr)间隔序列产生了315个匹配。经注释后获得了128个ORF和135个匹配的功能信息,占比前5位的依次为终止酶(terminase)、衣壳蛋白(capsid protein)、门蛋白(portal protein)、肽酶(peptidase)和DNA甲基化转移酶(DNA methyltransferase)。匹配多在病毒特定功能基因的保守域或关键结构域中。这表明,crispr-Cas系统发挥免疫功能时表现出针对病毒特定基因、功能和结构域的靶标特异性。此外,属于Class 1门类下的Type_I型系统的crispr-Cas间隔序列匹配数量远高于其他类型系统,占总数的89.0%。结论本文的研究结果揭示了crispr-Cas系统的靶标偏好,增进了对其的认识,为更好地理解病毒-宿主免疫互作机制提供了新的证据和线索。

关键词： crispr 间隔序列功能基因 DNA病毒

转座子编辑技术与crispr编辑技术在乳酸菌定向改良中应用的研究进展

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食品科学技术学报 2022年第6期40卷 134-144页

作者：杨勇艾连中熊智强杨昳津宋馨上海理工大学健康科学与工程学院/上海食品微生物工程技术研究中心上海200093

乳酸菌具有降胆固醇、维持菌群平衡、增强免疫等多种益生特性,是一类重要的食品工业微生物,而菌种改良经常用于改善工业上重要的微生物性能和功能。概述了传统改良、转座子诱变和crispr编辑3种乳酸菌改良技术的特点与应用。传统的乳酸... 详细信息

乳酸菌具有降胆固醇、维持菌群平衡、增强免疫等多种益生特性,是一类重要的食品工业微生物,而菌种改良经常用于改善工业上重要的微生物性能和功能。概述了传统改良、转座子诱变和crispr编辑3种乳酸菌改良技术的特点与应用。传统的乳酸菌改良技术包括适应性进化、随机诱变和原生质体融合,这些技术虽能改良乳酸菌,但操作复杂且收效低;转座子诱变是基于阻断转录或翻译过程而导致基因失活的一项技术,常被用于乳酸菌突变体库的构建,该技术高效简便,但依赖DNA链断裂、安全性低、不适用于广泛的宿主特性;crispr编辑技术在乳酸菌改良中应用广泛,可实现多位点同时编辑,是基因沉默和转录调控的强大工具,但该技术依赖DNA链断裂和同源重组,脱靶率较高。阐述了目前新出现的crispr相关转座子编辑系统的构建进行。crispr相关转座子编辑系统可实现供体DNA大片段(>10 kbp)的高效、精准、无标记编辑,且不依赖DNA双链断裂和同源重组,具有极低的脱靶率,在精准靶向和高效整合技术中显示出了极大优势,为未来乳酸菌精准高效的基因编辑技术提供了参考。希望可以为后续的乳酸菌遗传改良与基因功能分析提供相关理论指导。

关键词：乳酸菌遗传改良转座子编辑 crispr 精准高效整合

crispr base editor treats premature-aging syndrome

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Signal Transduction and Targeted Therapy 2021年第5期6卷 1410-1412页

作者： Ping Lin Jianxin Jiang Min Wu Wound Trauma Medical Center State Key Laboratory of TraumaBurns and Combined InjuryDaping HospitalArmy Medical UniversityChongqingChina Department of Biomedical Sciences School of Medicine and Health SciencesUniversity of North DakotaGrand ForksNDUSA

A recent paper published in Nature by Koblan et al.reported the use of crispr-mediated adenine base editor(ABE)to repair mutations of the Hutchinson-Gilford progeria syndrome(HGPS or progeria),attenuate symptoms,and extend lifespan of mice(Fig.1),representing a major advance in design of treatments for human accelerated-ageing disorders and potentially other genetic diseases.

关键词： crispr editor base

crispr单管等温扩增技术高灵敏检测核酸:以检测新型冠状病毒(SARS-CoV-2)RNA为例

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中国科学：化学 2022年第9期52卷 1685-1698页

作者：冯薇肖航袁爱姣邓兆佳王小燕彭汉勇乐晓春中国科学院生态环境研究中心环境化学与生态毒理学国家重点实验室北京100085 阿尔伯塔大学医学院病理和实验医学系分析和环境毒理研究所阿尔伯塔T6G2G3加拿大

簇状规则间隔短回文重复序列(crispr)及其相关蛋白(Cas)系统在分子检测领域正在快速发展,在核酸识别上已展现出高特异性、可编程性和多系统兼容性,成为一种极具潜力的分子诊断工具.单独crispr-Cas系统检测RNA的灵敏度受到其反式切割动... 详细信息

簇状规则间隔短回文重复序列(crispr)及其相关蛋白(Cas)系统在分子检测领域正在快速发展,在核酸识别上已展现出高特异性、可编程性和多系统兼容性,成为一种极具潜力的分子诊断工具.单独crispr-Cas系统检测RNA的灵敏度受到其反式切割动力学限制,因此需要与等温核酸扩增技术联用.将两个体系整合在单管中反应有两种策略:(1)将等温扩增与crispr-Cas体系分隔在管内不同位置进行分步反应;(2)在均相混合液中完成核酸扩增与检测.单管检测的关键是对多个反应的兼容性进行研究,从而最大限度地提升两种体系在同一条件下的性能,最后实现超高灵敏、快速、便捷地检测核酸.本文聚焦于基于crispr的单管超灵敏SARS-CoV-2 RNA检测技术,从方法开发的原理、性能和挑战等多个方面综述了近期的研究进展和发展方向,并指出理解crispr-Cas系统的反式切割动力学的重要性.

关键词： crispr 分子诊断 SARS-CoV-2 等温扩增酶反应动力学即时检测核酸

crispr base editing and prime editing:DSB and template-free editing systems for bacteria and plants

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Synthetic and Systems Biotechnology 2020年第4期5卷 277-292页

作者： Abdullah Zhengzheng Jiang Xulin Hong Shun Zhang Ruilian Yao Yi Xiao State Key Laboratory of Microbial Metabolism School of Life Sciences and BiotechnologyShanghai Jiao Tong UniversityShanghai200240China Joint International Research Laboratory of Metabolic&Developmental Sciences Shanghai Jiao Tong UniversityShanghai200240China

crispr-Cas(Clustered Regularly Interspaced Short Palindromic Repeats-crispr associated)has been extensively exploited as a genetic tool for genome editing.The RNA guided Cas nucleases generate DNA doublestrand break(DSB),triggering cellular repair systems mainly Non-homologous end-joining(NHEJ,imprecise repair)or Homology-directed repair(HDR,precise repair).However,DSB typically leads to unexpected DNA changes and lethality in some organisms.The establishment of bacteria and plants into major bio-production platforms require efficient and precise editing tools.Hence,in this review,we focus on the non-DSB and template-free genome editing,i.e.,base editing(BE)and prime editing(PE)in bacteria and plants.We first highlight the development of base and prime editors and summarize their studies in bacteria and plants.We then discuss current and future applications of BE/PE in synthetic biology,crop improvement,evolutionary engineering,and metabolic engineering.Lastly,we critically consider the challenges and prospects of BE/PE in PAM specificity,editing efficiency,off-targeting,sequence specification,and editing window.

关键词： crispr Base editing Prime editing Cytosine base editor Adenine base editor

Robust Transcriptional Activation in Plants Using Multiplexed crispr-Act2.0 and mTALE-Act Systems

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Molecular Plant 2018年第2期11卷 245-256页

作者： Levi G. Lowder Jianping Zhou Yingxiao Zhang Aimee Malzahn Zhaohui Zhong Tzung-Fu Hsieh Daniel F. Voytas Yong Zhang Yiping Qi Department of Biology East Carolina University Greenville NC 27858 USA Department of Biotechnology School of Life Science and Technology Center for informational Biology University of Electronic Science and Technology of ChinaChengdu 610054 China Department of Plant Science and Landscape Architecture University of Maryland College Park MD 20742 USA Department of Plant and Microbial Biology and Plants for Human Health Institute North Carolina State University North Carolina Research Campus KannapolisNC 28O81 USA Department of Genetics Ceil Biology and Development Center for Genome Engineering University of Minnesota Minneapolis MN 55455 USA Institute for Bioscience and Biotechnology Research University of Maryland Rockville MD 20850 USA

User-friendly tools for robust transcriptional activation of endogenous genes are highly demanded in plants. We previously showed that a dCas9-VP64 system consisting of the deactivated crispr- associated protein 9 （dCasg） fused with four tandem repeats of the transcriptional activator VP16 0/1=64） could be used for transcriptional activation of endogenous genes in plants. In this study, we developed a second generation of vector systems for enhanced transcriptional activation in plants. We tested multiple strategies for dCasg-based transcriptional activation, and found that simultaneous recruitment of VP64 by dCas9 and a modified guide RNA scaffold gRNA2.0 （designated crispr-Act2.0） yielded stronger transcrip- tional activation than the dCas9-VP64 system. Moreover, we developed a multiplex transcription activator- likeeffector activation （mTALE-Act） system for simultaneous activation of up to four genes in plants. Our results suggest that mTALE-Act is even more effective than crispr-Act2.0 in most cases tested. In addition, we explored tissue-specific gene activation using positive feedback loops. Interestingly, our study revealed that certain endogenous genes are more amenable than others to transcriptional activation, and tightly regulated genes may cause target gene silencing when perturbed by activation probes. Hence, these new tools could be used to investigate gene regulatory networks and their control mechanisms. Assembly of multiplex crispr-Act2.0 and mTALE-Act systems are both based on streamlined and PCR-independent Golden Gate and Gateway cloning strategies, which will facilitate transcriptional activation applications in both dicots and monocots.

关键词： crispr gRNA2.0 MS2-VP64 TALE-VP64 multiplex transcriptional activation

crispr technology is revolutionizing the improvement of tomato and other fruit crops

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Horticulture Research 2019年第1期6卷 781-793页

作者： Tian Wang Hongyan Zhang Hongliang Zhu College of Food Science and Nutritional Engineering China Agricultural University100083 BeijingChina Key Laboratory of Food Nutrition and Safety of Shandong Normal University College of Life ScienceShandong Normal University250014 JinanChina

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.

关键词： crispr crops breeding

An inducible crispr-ON system for controllable gene activation in human pluripotent stem cells

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Protein & Cell 2017年第5期8卷 379-393页

作者： Jianying Guo Dacheng Ma Rujin Huang Jia Ming Min Ye Kehkooi Kee Zhen Xie Jie Na Department of Basic Medical Sciences School of Medicine Center for Stem Cell Biology Tsinghua University Beijing 100084 China MOE Key Laboratory of Bioinformatics and Bioinformatics Division Center for Synthetic and System Biology TNLIST/ Department of Automation Tsinghua University Beijing 100084 China

Human pluripotent stem cells （hPSCs） are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a crispr.ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline （Dox） inducible dCasg-VP64-p65-Rta （dCas9-VPR） transcription activator and a reverse Tet transactivator （rtTA） expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by the addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG pro- moter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level promoted naive pluripotsnt gene expression, enhanced cell survival and clonogenicity, and enabled hESCs to integrate with the inner cell mass （ICM） of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs.

关键词： crispr transcription activation humanpluripotent stem cells NANOG pluripotency

crispr-mediated gene editing to rescue haploinsufficient obesity syndrome

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Protein & Cell 2019年第10期10卷 705-708页

作者： Zhifeng Wang Liu Yang Shen Qu Chao Zhang Translational Medical Center for Stem Cell Therapy and Institute for Regenerative Medicine Shanghai East HospitalShan ghai Key Laboratory of Signaling and Disease ResearchSchool of Life Sciences and Technologylongji UniversityShanghai 200092China Research&Development Department Sinoneural Cell and Gene Engineering Holdings Co.Ltd.ShanghaiChina Department of Endocri no logy and Metabolism National Metabolic Management CenterShanghai Tenth People's HospitalSchool of MedicineTongji UniversityShanghai 200092China

crispr and adeno-associated virus are becoming powerful tools to remedy genetic disorders in somatic cells of adulthood.A recent study published in Science(Matharu et al.,2019)safely targeted the non-coding genomic region of Sim1 and MC4R with rAAV packed with crispra,and successfully rescued the obesity syndrome caused by hap?loinsufficiency in a murine model,which shed light on their potential therapeutic applications in the future(Matharu et al.,2019).

关键词： crispr packed becoming