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Genome-wide crispr screens identify CLC-2 as a drug target for anti-herpesvirus therapy:tackling herpesvirus drug resistance

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Science China(Life Sciences) 2025年第2期68卷 515-526页

作者： Fayu Yang Nan Wei Shuo Cai Jing Liu Qingping Lan Hao Zhang Lu Shang Bo Zheng Mi Wang Yingchun Liu Lifang Zhang Chenzhong Fei Wu Tong Changlong Liu Ersheng Kuang Guangzhi Tong Feng Gu Shanghai Veterinary Research Institute Chinese Academy of Agricultural SciencesShanghai 200241China Key Laboratory of Veterinary Chemical Drugs and Pharmaceutics Ministry of Agriculture and Rural AffairsShanghai 200241China State Key Laboratory for Animal Disease Control and Prevention College of Veterinary MedicineLanzhou UniversityLanzhou Veterinary Research InstituteChinese Academy of Agricultural SciencesLanzhou 730000China Institute of Human Virology Zhongshan School of MedicineSun Yat-Sen UniversityGuangzhou 510080China School of Medicine Tsinghua UniversityBeijing 100084China

The emergence of drug resistance to virus(i.e.,acyclovir(ACV)to herpesviruses)has been termed one of the common clinical issues,emphasizing the discovery of new antiviral *** address it,a genome-wide clustered regularly interspaced short palindromic repeats(crispr)screening was performed in mouse haploid embryonic stem cells infected with pseudorabies virus(PRV),anα-herpesvirus causing human and pig *** results demonstrated that type 2 voltage-gated chloride channels(CLC-2)encoded by one of the identified genes,CLCN2,is a potential drug target for anti-herpesvirus ***-2 inhibitors,omeprazole(OME)and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid(DIDS),can efficiently inhibit infection of multiple herpesviruses in cellulo(i.e.,PRV,HSV and EBV),and effectively treat murine herpes simplex encephalitis(HSE).Additionally,DIDS was found to inhibit HSV-1 replication by blocking the PI3K/Akt *** importantly,both DIDS and OME were able to inhibit ACV-resistant HSV-1 strain *** study's findings suggest that targeting host-cell factors such as CLC-2 may be a promising approach to tackling herpesvirus drug *** discovery of CLC-2 as a potential drug target for anti-herpesvirus therapy provides a new direction for the development of novel antiviral agents.

关键词： herpesviruses crispr host-directed therapy CLC-2 DIDS

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基于one-pot RPA-crispr/Cas12a技术快速检测高毒力肺炎克雷伯杆菌方法的建立

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微生物学通报 2025年

作者：段红雯李彩燕朱鹏张顺浙江万里学院生物与环境学院国科宁波生命与健康产业研究院宁波市第二医院

【背景】在肺部健康红灯预警的社会背景下，高毒力肺炎克雷伯杆菌（hypervirulent Klebsiella Pneumoniae， hvKP）的出现引起了广泛关注。作为经典肺炎克雷伯杆菌的变种，hvKP有易传播、毒性强、致死率高的特点，易在健康人中引发社区... 详细信息

【背景】在肺部健康红灯预警的社会背景下，高毒力肺炎克雷伯杆菌（hypervirulent Klebsiella Pneumoniae， hvKP）的出现引起了广泛关注。作为经典肺炎克雷伯杆菌的变种，hvKP有易传播、毒性强、致死率高的特点，易在健康人中引发社区获得性感染。【目的】开发出针对于hvKP的灵敏便捷的检测方法预防其大面积的传播成为了重中之重的工作。【方法】将重组酶聚合酶等温扩增与crispr超灵敏检测系统相结合，开发出一种针对hvKP的便捷且高灵敏的检测方法。【结果】该方法在无增菌的条件下能够在40 min内检测低至58 fg/μL的hvKP的DNA样本，且在特异性评估中表现出色，在13种非特异性菌种包括经典肺炎克雷伯杆菌均无扩增，显示了良好的特异性。此外，在模拟血液样本的检测中，该方法的灵敏度达到5.5 CFU/mL，高于传统的qRCR方法(灵敏度为5.5×102CFU/mL)两个数量级。【结论】该新型检测方法不仅具有高灵敏度和高特异性的优点，而且因其构建方法简便，检测过程便捷，旨在为基层医疗机构大规模筛查hvKP提供有效工具，同时也为公共卫生服务领域提供新的检测方案。这一新技术有望在控制和预防hvKP传播方面发挥重要作用。

关键词：高毒力肺炎克雷伯杆菌 crispr Cas12a 重组酶聚合酶扩增

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基于crispr的细胞产品的临床应用及监管挑战

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中国医药导刊 2025年第1期27卷 101-108页

作者：赵宝龙华坚张长风上海医药集团生物治疗技术有限公司上海200072

以嵌合抗原受体T细胞(CAR-T)为代表的免疫细胞治疗产品为多种癌症的治疗策略带来了突破性改变,但实体瘤疗效以及异体使用等问题仍阻碍细胞治疗领域的进一步发展。基因编辑技术可以通过改造免疫细胞的生物学功能来克服上述技术瓶颈。其中... 详细信息

以嵌合抗原受体T细胞(CAR-T)为代表的免疫细胞治疗产品为多种癌症的治疗策略带来了突破性改变,但实体瘤疗效以及异体使用等问题仍阻碍细胞治疗领域的进一步发展。基因编辑技术可以通过改造免疫细胞的生物学功能来克服上述技术瓶颈。其中,规律间隔成簇短回文重复序列(crispr)技术由于具备成本低、准确度高等特点而成为目前最常用的基因编辑工具。因此,crispr也被认为是开发下一代细胞治疗产品的关键。然而,作为一种新兴技术,crispr会在细胞产品中诱导非预期的基因改变,并对临床应用带来潜在风险,而行业内对此类风险的了解仍非常有限。本研究结合细胞治疗领域的最新研究情况,概述crispr技术在细胞治疗产品中的研究进展,总结2016—2024年全球开展的基于crispr技术的细胞产品的临床试验情况,重点描述临床研究类型、基因编辑靶点、适应证种类等要素,并基于国内外政策法规与临床实践情况,分析crispr技术相关细胞产品的基因风险以及评估策略,以期为该类产品的临床开发和监管政策制定提供借鉴。

关键词： crispr 基因编辑细胞治疗药品监管监管挑战

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crispr/Cas9: Development and Application in Rice Breeding

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Rice science 2019年第5期26卷 265-281页

作者： Fernando Matías ROMERO Andrés GATICA-ARIAS Laboratory of Plant Biotechnology School of Biology/Research Center in Cellular and Molecular Biology University of Costa Rica

Rice(Oryza sativa L.)is an important staple food crop worldwide due to its adaptability to different environmental *** of its great economic and social importance,there is a constant requirement for new varieties with improved agronomic characteristics,such as tolerance to different biotic(such as bacterium,fungus,insect and virus)and abiotic stresses(such as salinity,drought and temperature),higher yield and better organoleptic and nutritional *** the new genome editing technologies,the clustered regularly interspaced short palindromic repeats(crispr)/crispr-associated protein(Cas)(crispr/Cas)system allows precise and specific edition in a targeted genome *** is one of the most frequently used techniques for the study of the function of new genes and for the development of mutant lines with enhanced tolerance to biotic and abiotic stresses,herbicide resistance or improved *** wide varieties of applications for this technology include simple non-homologous end joining,homologous recombination,gene replacement,and base *** this review,we analyzed how some of these applications have been used in rice cultivars to obtain rice varieties better adapted to current environmental conditions and market requirements.

关键词： rice crispr Cas genome editing biotic stress abiotic stress yield off-target mutation

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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 *** CD processes governing genetic flow include the cell cycle,DNA replication,chromosome packaging,epigenetic changes,transcription,posttranscriptional alterations,translation,and posttranslational *** CD processes are tightly regulated in plants to maintain genetic integrity throughout the life cycle and to pass genetic materials to next *** of various CD processes involved in gene regulation will accelerate crop improvement to feed the growing world *** technology enables programmable editing of CD processes to alter DNA,RNA,or protein,which would have been impossible in the ***,an overview of recent advancements in crispr tool development and crispr-based CD modulations that expedite basic and applied plant research is ***,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 ***,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

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crispr-assisted transcription activation by phaseseparation proteins

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Protein & Cell 2023年第12期14卷 874-887页

作者： Jiaqi Liu Yuxi Chen Baoting Nong Xiao Luo Kaixin Cui Zhan Li Pengfei Zhang Wenqiong Tan Yue Yang Wenbin Ma Puping Liang Zhou Songyang State Key Laboratory of Biocontrol MOE Key Laboratory of Gene Function and Regulation and Guangzhou Key Laboratory of Healthy Aging ResearchSchool of Life SciencesSun Yat-senUniversityGuangzhou510275China Sun Yat-sen Memorial Hospital Sun Yat-sen UniversityGuangzhou 510275China Lumiere Therapeutics Suzhou215000China

The clustered regularly interspaced short palindromic repeats(crispr)-Cas9 system has been widely used for genome engineering and transcriptional regulation in many different *** crispr-activation(crispra)platforms often require multiple components because of inefficient transcriptional ***,we fused different phase-separation proteins to dCas9-VPR(dCas9-VP64-P65-RTA)and observed robust increases in transcriptional activation ***,human NUP98(nucleoporin 98)and FUS(fused in sarcoma)IDR domains were best at enhancing dCas9-VPR activity,with dCas9-VPR-FUS IDR(VPRF)outperforming the other crispra systems tested in this study in both activation efficiency and system ***9-VPRF overcomes the target strand bias and widens gRNA designing windows without affecting the off-target effect of *** findings demonstrate the feasibility of using phase-separation proteins to assist in the regulation of gene expression and support the broad appeal of the dCas9-VPRF system in basic and clinical applications.

关键词： crispr transcriptional activation phase-separation proteins

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crispr-Cas13-mediated RNA editing in the silkworm Bombyx mori

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Zoological Research 2024年第6期45卷 1249-1260页

作者： Yao-Hao Tang Xing Zhang Zong-Cai Dai Hao Li Yan Yang Tu-Jing Zhao Dong-Qin Yuan Wen-Liang Qian Dao-Jun Cheng Integrative Science Center of Germplasm Creation in Western China(CHONGQING)Science City Biological Science Research CenterSouthwest UniversityChongqing 400715China State Key Laboratory of Resource Insects Southwest UniversityChongqing 400715China

The crispr-Cas13 system,an RNA-guided editing tool,has emerged as a highly efficient and stable RNA editing *** the crispr-Cas13 system has been developed in several insect species,its application in lepidopterans has not yet been *** the present study,we evaluated the RNA cleavage activity of the crispr-Cas13 system in the silkworm(Bombyx mori),a model lepidopteran insect,both ex vivo and in *** established two stable silkworm BmE cell lines expressing PspCas13b and CasRx,*** analysis demonstrated that both PspCas13b and CasRx effectively down-regulated the transcription of exogenouslyintroduced target and endogenous genes in these cell *** addition,we generated two transgenic silkworm strains,one expressing CasRx and the other expressing RNA-guided crispr RNA targeting Sex combs reduced(Scr).Further crossing experiments showed that CasRx induced a down-regulation of Scr transcription in silkworms,which impaired systemic growth of ***,this study demonstrated that the crispr-Cas13RNA editing system works efficiently in the silkworm,providing a potential alternative approach for RNA manipulation in lepidopteran insects.

关键词： Silkworm crispr PspCas13b CasRx RNA editing

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crispr,CAR-T,and NK:Current applications and future perspectives

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Genes & Diseases 2024年第4期11卷 246-257页

作者： Mohadeseh Khoshandam Hossein Soltaninejad Amir Ali Hamidieh Saman Hosseinkhani Department of Reproductive Biology Academic Center for EducationCultureand Research(ACECR)Qom branch***Iran National Institute of Genetic Engineering and Biotechnology(NIGEB) Tehran 14965/161Iran Department of stem cells technology and Tissue Regeneration Faculty of Interdisciplinary Science and TechnologiesTarbiat Modares UniversityTehran 15614Iran Pediatric Cell Therapy and Gene Therapy Research Center GeneCell&Tissue Research InstituteTehran University of Medical SciencesTehran ***Iran Department of Biochemistry Faculty of Biological SciencesTarbiat Modares UniversityTehran 15614Iran

Chimeric antigen receptor T(CAR-T)cell therapy represents a breakthrough in personalized cancer *** this regard,synthetic receptors comprised of antigen recognition domains,signaling,and stimulatory domains are used to reprogram T-cells to target tum or cells and destroy *** the success of this approach in refractory B-cell malignancies,the optimal potency of CAR T-cell therapy for many other cancers,particularly solid tumors,has not been *** killer cells are powerful cytotoxic lymphocytes specialized in recognizing and dispensing the tumor cells in coordination with other anti-tumor immunity *** on these studies,many investigations are focused on the accurate designing of CAR T-cells with clustered regularly interspaced short palindromic repeats(crispr)/crispr-associated protein 9(Cas9)system or other novel gene editing tools that can induce hereditary changes with or without the presence of a double-stranded break into the *** methodologies can be specifically focused on negative controllers of T-cells,induce modifications to a particular gene,and produce reproducible,safe,and powerful allogeneic CAR T-cells for on-demand cancer *** improvement of the crispr/Cas9 innovation offers an adaptable and proficient gene-editing capability in activating different pathways to help natural killer cells interact with novel CARs to particularly target tumor *** achievements and future challenges of combining next-generation crispr-Cas9 gene editing tools to optimize CAR T-cell and natural killer cell treatment for future clinical trials toward the foundation of modern cancer treatments have been assessed inthis review.

关键词： Cancer CAR T-cell crispr Gene editing Immunotherapy NK cell

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crispr-Based Genome-Editing Tools for Huntington’s Disease Research and Therapy

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Neuroscience Bulletin 2022年第11期38卷 1397-1408页

作者： Yiyang Qin Shihua Li Xiao-Jiang Li Su Yang Guangdong Key Laboratory of Non-human Primate Research Guangdong-Hongkong-Macao Institute of CNS RegenerationJinan UniversityGuangzhou 510632China

Huntington’s disease(HD)is an autosomal dominantly-inherited neurodegenerative disease,which is caused by CAG trinucleotide expansion in exon 1 of the Huntingtin(HTT)*** HD is a rare disease,its monogenic nature makes it an ideal model in which to understand pathogenic mechanisms and to develop therapeutic strategies for neurodegenerative *** regularly-interspaced short palindromic repeats(crispr)is the latest technology for genome *** simple to use and highly efficient,crispr-based genome-editing tools are rapidly gaining popularity in biomedical research and opening up new avenues for disease ***,we review the development of crispr-based genome-editing tools and their applications in HD research to offer a translational perspective on advancing the genome-editing technology to HD treatment.

关键词： Huntington’s disease crispr Animal models

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A crispr/Cas12a-empowered surface plasmon resonance platform for rapid and specific diagnosis of the Omicron variant of SARS-CoV-2

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National Science Review 2022年第8期9卷 100-109页

作者： Zhi Chen Jingfeng Li Tianzhong Li Taojian Fan Changle Meng Chaozhou Li Jianlong Kang Luxiao Chai Yabin Hao Yuxuan Tang Omar A.Al-Hartomy Swelm Wageh Abdullah G.Al-Sehemi Zhiguang Luo Jiangtian Yu Yonghong Shao Defa Li Shuai Feng William J.Liu Yaqing He Xiaopeng Ma Zhongjian Xie Han Zhang Shenzhen Engineering Laboratory of Phosphorene and Optoelectronics International Collaborative Laboratory of 2D Materials for Optoelectronics Science and Technology of Ministry of Education Shenzhen Institute of Translational Medicine Department of Otolaryngology Shenzhen Second People’s Hospital the First Affiliated Hospital Institute of Microscale Optoelectronics Shenzhen University Shenzhen International Institute for Biomedical Research Shenzhen Han’s Tech Limited Company Shenzhen Metasensing Tech Limited Company Department of Physics Faculty of Science King Abdulaziz University Research Center for Advanced Materials Science (RCAMS) King Khalid University Department of Chemistry College of Science King Khalid University Zhongmin (Shenzhen) Intelligent Ecology Co.Ltd Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province College of Physics and Optoelectronic Engineering Shenzhen University Department of Laboratory Medicine Shenzhen Children’s Hospital Optoelectronics Research Center School of Science Minzu University of China NHC Key Laboratory of Biosafety National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention Research Unit of Adaptive Evolution and Control of Emerging Viruses Chinese Academy of Medical Sciences Institute of Pathogenic Organism Shenzhen Center for Disease Control and Prevention Respiratory Department Shenzhen Children’s Hospital Institute of Pediatrics Shenzhen Children’s Hospital

The outbreak of the COVID-19 pandemic was partially due to the challenge of identifying asymptomatic and presymptomatic carriers of the virus, and thus highlights a strong motivation for diagnostics with high sensitivity that can be rapidly deployed. On the other hand, several concerning SARS-Co V-2 variants,including Omicron, are required to be identified as soon as the samples are identified as ‘positive'.Unfortunately, a traditional PCR test does not allow their specific identification. Herein, for the first time,we have developed MOPCS(Methodologies of Photonic crispr Sensing), which combines an optical sensing technology-surface plasmon resonance(SPR) with the ‘gene scissors' clustered regularly interspaced short palindromic repeat(crispr) technique to achieve both high sensitivity and specificity when it comes to measurement of viral variants. MOPCS is a low-cost, crispr/Cas12a-systemempowered SPR gene-detecting platform that can analyze viral RNA, without the need for amplification,within 38 min from sample input to results output, and achieve a limit of detection of 15 fM. MOPCS achieves a highly sensitive analysis of SARS-Co V-2, and mutations appear in variants B.1.617.2(Delta),B.1.1.529(Omicron) and BA.1(a subtype of Omicron). This platform was also used to analyze some recently collected patient samples from a local outbreak in China, identified by the Centers for Disease Control and Prevention. This innovative crispr-empowered SPR platform will further contribute to the fast, sensitive and accurate detection of target nucleic acid sequences with single-base mutations.

关键词： surface plasmon resonance crispr SARS-CoV-2 Omicron variant

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