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crispr基因组编辑技术在传染病致病机理研究和防诊治中的应用

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遗传 2023年第11期45卷 950-962页

作者：杨臻嵘周钢桥中国科学技术大学生命科学学院生命科学与医学部合肥230022 军事科学院军事医学研究院辐射医学研究所医学蛋白质组全国重点实验室国家蛋白质科学中心北京100850

crispr基因组编辑技术在基因操作和传染病研究等方面展现出巨大的应用前景,对于有效控制和治愈传染病具有重要价值。通过其构建的细胞、类器官和动物疾病模型,为探索传染病相关分子机制提供了极大便利。crispr筛选技术使得高通量鉴定传... 详细信息

crispr基因组编辑技术在基因操作和传染病研究等方面展现出巨大的应用前景,对于有效控制和治愈传染病具有重要价值。通过其构建的细胞、类器官和动物疾病模型,为探索传染病相关分子机制提供了极大便利。crispr筛选技术使得高通量鉴定传染病相关风险因子成为可能。基于crispr的新型分子诊断工具为病原体的检测提供了更灵敏和快速的方法。利用crispr工具敲入抗性基因或破坏风险基因和病毒基因组,有望实现预防或治疗传染病。本综述讨论了crispr基因组编辑技术在疾病模型制备、传染病风险因子筛选、病原体诊断和传染病防治中的应用,以期为后续传染病的研究和防诊治提供参考。

关键词：传染病 crispr 致病机理诊断防治

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基于crispr的快速灵敏便捷分子检测

生物工程学报 2023年第1期39卷 60-73页

作者：孙雯君黄行许王鑫杰上海科技大学生命科学与技术学院上海201210 中国农业科学院(深圳)农业基因组研究所、岭南现代农业科学与技术广东省实验室深圳分中心、农业农村部农业基因数据分析重点实验室广东深圳518120 广州生物岛实验室广东广州510030

快速灵敏检测技术对疾病防控必不可少。特别是新冠疫情暴发以来,人们深刻认识到快速灵敏检测技术的重要性。近年来,以crispr/Cas为代表的基因编辑技术带来了生物技术革命性的进步。crispr的核酸检测技术因其快速、准确、灵敏、经济等特... 详细信息

快速灵敏检测技术对疾病防控必不可少。特别是新冠疫情暴发以来,人们深刻认识到快速灵敏检测技术的重要性。近年来,以crispr/Cas为代表的基因编辑技术带来了生物技术革命性的进步。crispr的核酸检测技术因其快速、准确、灵敏、经济等特点,正在引发分子诊断革新,并已被成功应用于传染病、遗传病、肿瘤基因突变诊断,以及食品安全等领域。本文归纳了基于crispr的多种核酸检测体系及应用,并对未来crispr核酸检测发展趋势及结合人工智能的智能化检测进行了展望。

关键词：分子诊断核酸检测临场检测基因编辑 crispr crispr检测

基于crispr/Cas12b的乳粉中克罗诺杆菌属检测

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中国食品学报 2022年第6期22卷 267-275页

作者：张懿翔张清平李林显王亮杨捷琳刘洋上海市质量监督检验技术研究院/国家市场监管重点实验室(乳及乳制品检测与监控技术) 上海200233 吐露港生物科技有限公司上海200233 上海海关动植物与食品检验检疫技术中心上海200135

目的:开发一种基于crispr/Cas12b的克罗诺杆菌属分子检测方法。方法:以克罗诺杆菌菌属特异性基因序列为候选区域,针对区域设计扩增引物和向导RNA靶向序列,对其进行条件优化、特异性检测、反应体系优化,并用人工污染样品对体系的灵敏度... 详细信息

目的:开发一种基于crispr/Cas12b的克罗诺杆菌属分子检测方法。方法:以克罗诺杆菌菌属特异性基因序列为候选区域,针对区域设计扩增引物和向导RNA靶向序列,对其进行条件优化、特异性检测、反应体系优化,并用人工污染样品对体系的灵敏度、重复性进行验证。结果:该方法的特异性很好,方法的灵敏度达0.01 ng/μL,在人工污染样品中可检测到初始菌量crispr/Cas12b方法可用于乳粉中克罗诺杆菌属的快速检测。

关键词： crispr Cas12b 克罗诺杆菌属检测

crispr ribonucleoprotein-mediated genetic engineering in plants

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Plant Communications 2021年第2期2卷 38-50页

作者： Yingxiao Zhang Brian Iaffaldano Yiping Qi Department of Plant Science and Landscape Architecture University of MarylandCollege ParkMD 20742USA Institute for Bioscience and Biotechnology Research University of MarylandRockvilleMD 20850USA

crispr-derived biotechnologies have revolutionized the genetic engineering field and have been widely applied in basic plant research and crop improvement.Commonly used Agrobacterium-or particle bombardment-mediated transformation approaches for the delivery of plasmid-encoded crispr reagents can result in the integration of exogenous recombinant DNA and potential off-target mutagenesis.Editing efficiency is also highly dependent on the design of the expression cassette and its genomic insertion site.Genetic engineering using crispr ribonucleoproteins(RNPs)has become an attractive approach with many advantages:DNA/transgene-free editing,minimal off-target effects,and reduced toxicity due to the rapid degradation of RNPs and the ability to titrate their dosage while maintaining high editing efficiency.Although RNP-mediated genetic engineering has been demonstrated in many plant species,its editing efficiency remains modest,and its application in many species is limited by difficulties in plant regeneration and selection.In this review,we summarize current developments and challenges in RNPmediated genetic engineering of plants and provide future research directions to broaden the use of this technology.

关键词： crispr RNP genetic engineering genome editing transgene free

crispr-PIN:Modifying gene position in the nucleus via dCas9-mediated tethering

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Synthetic and Systems Biotechnology 2019年第2期4卷 73-78页

作者： Jyun-Liang Lin Holly Ekas Matthew Deaner Hal S.Alper McKetta Department of Chemical Engineering The University of Texas at Austin200 E Dean Keeton St.Stop C0400AustinTX 78712USA Institute for Cellular and Molecular Biology The University of Texas at Austin2500 Speedway AvenueAustinTX 78712USA

Spatial organization of DNA within the nucleus is important for controlling DNA replication and repair,genetic recombination,and gene expression.Here,we present crispr-PIN,a crispr/dCas9-based tool that allows control of gene Position in the Nucleus for the yeast Saccharomyces cerevisiae.This approach utilizes a cohesindockerin interaction between dCas9 and a perinuclear protein.In doing so,we demonstrate that a single gRNA can enable programmable interaction of nuclear DNA with the nuclear periphery.We demonstrate the utility of this approach for two applications:the controlled segregation of an acentric plasmid and the re-localization of five endogenous loci.In both cases,we obtain results on par with prior reports using traditional,more cumbersome genetic systems.Thus,crispr-PIN offers the opportunity for future studies of chromosome biology and gene localization.

关键词： crispr Chromosome organization Chromosome biology Gene positioning Synthetic biology

crispr-Mediated Engineering across the Central Dogma in Plant Biology for Basic Research and Crop Improvement

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Molecular Plant 2021年第1期14卷 127-150页

作者： Dibyajyoti Pramanik Rahul Mahadev Shelake Mi Jung Kim Jae-Yean Kim Division of Applied Life Science(BK21 Four Program) Plant Molecular Biology and Biotechnology Research CenterGyeongsang National UniversityJinju 52828Korea These authors contributed equally to this article

The central dogma(CD)of molecular biology is the transfer of genetic information from DNA to RNA to protein.Major CD processes governing genetic flow include the cell cycle,DNA replication,chromosome packaging,epigenetic changes,transcription,posttranscriptional alterations,translation,and posttranslational modifications.The CD processes are tightly regulated in plants to maintain genetic integrity throughout the life cycle and to pass genetic materials to next generation.Engineering of various CD processes involved in gene regulation will accelerate crop improvement to feed the growing world population.crispr technology enables programmable editing of CD processes to alter DNA,RNA,or protein,which would have been impossible in the past.Here,an overview of recent advancements in crispr tool development and crispr-based CD modulations that expedite basic and applied plant research is provided.Furthermore,crispr applications in major thriving areas of research,such as gene discovery(allele mining and cryptic gene activation),introgression(de novo domestication and haploid induction),and application of desired traits beneficial to farmers or consumers(biotic/abiotic stress-resilient crops,plant cell factories,and delayed senescence),are described.Finally,the global regulatory policies,challenges,and prospects for crispr-mediated crop improvement are discussed.

关键词： central dogma crispr crop improvement gene regulation genome editing

crispr-Cas nucleases and base editors for plant genome editing

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aBIOTECH 2020年第1期1卷 74-87页

作者： Filiz Gurel Yingxiao Zhang Simon Sretenovic Yiping Qi Department of Molecular Biology and Genetics Faculty of ScienceIstanbul UniversityVezneciler34134 IstanbulTurkey Department of Plant Science and Landscape Architecture University of MarylandCollege ParkMD 20742USA Institute for Bioscience and Biotechnology Research University of MarylandRockvilleMD 20850USA

Clustered regularly interspaced short palindromic repeats(crispr)—crispr-associated protein(Cas)and base editors are fundamental tools in plant genome editing.Cas9 from Streptococcus pyogenes(SpCas9),recognizing an NGG protospacer adjacent motif(PAM),is a widely used nuclease for genome editing in living cells.Cas12a nucleases,targeting T-rich PAMs,have also been recently demonstrated in several plant species.Furthermore,multiple Cas9 and Cas12a engineered variants and orthologs,with different PAM recognition sites,editing efficiencies and fidelity,have been explored in plants.These RNA-guided sequence-specific nucleases(SSN)generate double-stranded breaks(DSBs)in DNA,which trigger non-homologous end-joining(NHEJ)repair or homology-directed repair(HDR),resulting in insertion and deletion(indel)mutations or precise gene replacement,respectively.Alternatively,genome editing can be achieved by base editors without introducing DSBs.So far,several base editors have been applied in plants to introduce C-to-T or A-to-G transitions,but they are still undergoing improvement in editing window size,targeting scope,off-target effects in DNA and RNA,product purity and overall activity.Here,we summarize recent progress on the application of Cas nucleases,engineered Cas variants and base editors in plants.

关键词： crispr SpCas9 Cas12a Cas12b PAM Cytidine/adenine base editors

基于分子信标策略的crispr/Cas12a荧光传感器用于循环肿瘤DNA的放大检测

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分析化学 2023年第2期51卷 184-193页

作者：张函笑代晓春周亚楠吕旭珍弓韬赵旭华于保锋山西医科大学基础医学院生物化学与分子生物学教研室

构建了一种以分子信标（Molecular beacon, MB）为信号探针的crispr/Cas12a生物传感器，用于循环肿瘤DNA(Circular tumor DNA, ctDNA)的快速放大检测。MB具有良好的稳定性，其颈部末端分别标记有荧光素（FAM）和四甲基罗丹明（TAMRA）... 详细信息

构建了一种以分子信标（Molecular beacon, MB）为信号探针的crispr/Cas12a生物传感器，用于循环肿瘤DNA(Circular tumor DNA, ctDNA)的快速放大检测。MB具有良好的稳定性，其颈部末端分别标记有荧光素（FAM）和四甲基罗丹明（TAMRA）两种荧光基团。ctDNA不存在时，crispr/Cas12a体系无活性，无法切割MB，因此MB两端的荧光基团由于形成发夹结构的颈部相互靠近而发生荧光共振能量转移（Fluorescence resonance energy transfer, FRET），显示出TAMRA的荧光。当ctDNA存在时，ctDNA特异性识别Cas12a/crRNA二元复合物并激活Cas12a的反式切割活性。由于单链DNA是Cas12a最敏感的底物，因此MB的环部单链首先被切割，继而引起颈部双链的解离而导致两种荧光团彼此远离，无法发生FRET，最终显示出FAM的荧光信号。对MB环部的碱基数目、MB浓度以及crRNA与Cas12a的浓度比例等实验条件进行了优化。在最优条件下，1.7～500 pmol/L范围内，ctDNA浓度与传感器在518 nm处的荧光强度呈线性关系，检出限为0.6 pmol/L(3σ)。将此传感器用于血清样品中ctDNA的检测，回收率在93%～110%之间。

关键词： crispr 生物传感器荧光共振能量转移循环肿瘤DNA

crispr Primer Designer:Design primers for knockout and chromosome imaging crispr-Cas system

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Journal of Integrative Plant Biology 2015年第7期57卷 613-617页

作者： Meng Yan Shi-Rong Zhou Hong-Wei Xue National Key Laboratory of Plant Molecular Genetics Shanghai Institute of Plant Physiology and Ecologythe Chinese Academy of Sciences

The clustered regularly interspaced short palin- dromic repeats （crispr）-associated system enables biologists to edit genomes precisely and provides a powerful tool for perturbing endogenous gene regulation, modulation of epigenetic markers, and genome architecture. However, there are concerns about the specificity of the system, especially the usages of knocking out a gene. Previous designing tools either were mostly built-in websites or ran as command-line programs, and none of them ran locally and acquired a user- friendly interface. In addition, with the development of crispr-derived systems, such as chromosome imaging, there were still no tools helping users to generate specific end-user spacers. We herein present researchers to design primers crispr Primer Designer for for crispr applications. Theprogram has a user-frfendly interface, can analyze the BLAST results by using multiple parameters, score for each candidate spacer, and generate the primers when using a certain plasmid. In addition, crispr Primer Designer runs locally and can be used to search spacer clusters, and exports primers for the crispr-Cas system-based chromosome imaging system.

关键词： Chromosome imaging crispr knockout primer software