CRISPR/Cas9-based genome editing has been one of the major achievements of molecular biology,allowing the targeted engineering of a wide range of genomes.The system originally evolved in prokaryotes as an adaptive imm...CRISPR/Cas9-based genome editing has been one of the major achievements of molecular biology,allowing the targeted engineering of a wide range of genomes.The system originally evolved in prokaryotes as an adaptive immune system against bacteriophage infections.It now sees widespread application in genome engineering workflows,especially using the Streptococcus pyogenes endonuclease Cas9.To utilize Cas9,so-called single guide RNAs(sgRNAs)need to be designed for each target gene.While there are many tools available to design sgRNAs for the popular model organisms,only few tools that allow designing sgRNAs for non-model organisms exist.Here,we present CRISPy-web(http://crispy.secondarymetabolites.org/),an easy to use web tool based on CRISPy to design sgRNAs for any userprovided microbial genome.CRISPy-web allows researchers to interactively select a region of their genome of interest to scan for possible sgRNAs.After checks for potential off-target matches,the resulting sgRNA sequences are displayed graphically and can be exported to text files.All steps and information are accessible from a web browser without the requirement to install and use command line scripts.展开更多
成簇的规律间隔的短回文重复序列及其相关蛋白9〔clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated protein 9(Cas9),CRISPR/Cas9〕是一种新兴的基因编辑技术,与以前的三大基因编辑技术——归巢核...成簇的规律间隔的短回文重复序列及其相关蛋白9〔clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated protein 9(Cas9),CRISPR/Cas9〕是一种新兴的基因编辑技术,与以前的三大基因编辑技术——归巢核酸内切酶、锌指核酸酶和转录激活因子样效应物核酸酶技术相比,其在靶向特异性、操作简便性、治疗彻底性、应用广泛性等方面具有更大的优势和发展潜力。艾滋病、乙型肝炎、疟疾等感染性疾病的治疗一直是医学上的重大难题,科学家正努力尝试利用CRISPR/Cas9技术解决这些医学难题。本文主要综述了CRISPR/Cas9技术在这些感染性疾病中应用的研究进展。展开更多
CRISPR/Cas9-mediated genome editing can inhibit virus infection by targeting the conserved regions of the viral genomic DNA. Unexpectedly, we found previously that pseudorabies virus(PRV) could escape from CRISPR/Cas9...CRISPR/Cas9-mediated genome editing can inhibit virus infection by targeting the conserved regions of the viral genomic DNA. Unexpectedly, we found previously that pseudorabies virus(PRV) could escape from CRISPR/Cas9-mediated inhibition.In order to elucidate whether the escape of PRV from Cas9-mediated inhibition was due to cell deficiencies, such as genetic instability of sgRNA or Cas9 protein, the positive cells were passaged ten times, and PRV infection in the sgRNA-expressing cells was evaluated in the present study. The results showed that subculturing cells has no effect on Cas9-mediated cleavage of PRV. Different passages of PX459-PRV cells can stably express sgRNA to facilitate Cas9/sgRNA cleavage on the UL30 gene of PRV, resulting in a pronounced inhibition of PRV infection. Studies to elucidate the mechanism of PRV escape are currently in progress.展开更多
BACKGROUND： Visualization of chromosomal loci location and dynamics is crucial for understanding many fundamental intra-nuclear processes such as DNA transcription, replication, and repair. OBJECTIVE： Here, we will ...BACKGROUND： Visualization of chromosomal loci location and dynamics is crucial for understanding many fundamental intra-nuclear processes such as DNA transcription, replication, and repair. OBJECTIVE： Here, we will describe the development of fluorescence labeling methods for chromatin imaging, including traditional as well as emerging chromatin labeling techniques in both fixed and live cells. We will also discuss current issues and provide a perspective on future developments and applications of the chromatin labeling technology. METHODS： A systematic literature search was performed using the PubMed. Studies published over the past 50 years were considered for review. More than 100 articles were cited in this review. RESULTS： Taking into account sensitivity, specificity, and spatiotemporal resolution, fluorescence labeling and imaging has been the most prevalent approach for chromatin visualization. Among all the fluorescent labeling tools, the adoption ofgenome editing tools, such as TALE and CRISPR, have great potential for the labeling and imaging of chromatin. CONCLUSION： Although a number of chromatin labeling techniques are available for both fixed and live cells, much more effort is still clearly required to develop fluorescence labeling methods capable of targeting arbitrary sequences non-intrusively to allow long-term, multiplexing, and high-throughput imaging of genomic loci and chromatin structures. The emerging technological advances will outline a next-generation effort toward the comprehensive delineation of chromatin at single-cell level with single-molecule resolution.展开更多
文摘CRISPR/Cas9-based genome editing has been one of the major achievements of molecular biology,allowing the targeted engineering of a wide range of genomes.The system originally evolved in prokaryotes as an adaptive immune system against bacteriophage infections.It now sees widespread application in genome engineering workflows,especially using the Streptococcus pyogenes endonuclease Cas9.To utilize Cas9,so-called single guide RNAs(sgRNAs)need to be designed for each target gene.While there are many tools available to design sgRNAs for the popular model organisms,only few tools that allow designing sgRNAs for non-model organisms exist.Here,we present CRISPy-web(http://crispy.secondarymetabolites.org/),an easy to use web tool based on CRISPy to design sgRNAs for any userprovided microbial genome.CRISPy-web allows researchers to interactively select a region of their genome of interest to scan for possible sgRNAs.After checks for potential off-target matches,the resulting sgRNA sequences are displayed graphically and can be exported to text files.All steps and information are accessible from a web browser without the requirement to install and use command line scripts.
文摘成簇的规律间隔的短回文重复序列及其相关蛋白9〔clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated protein 9(Cas9),CRISPR/Cas9〕是一种新兴的基因编辑技术,与以前的三大基因编辑技术——归巢核酸内切酶、锌指核酸酶和转录激活因子样效应物核酸酶技术相比,其在靶向特异性、操作简便性、治疗彻底性、应用广泛性等方面具有更大的优势和发展潜力。艾滋病、乙型肝炎、疟疾等感染性疾病的治疗一直是医学上的重大难题,科学家正努力尝试利用CRISPR/Cas9技术解决这些医学难题。本文主要综述了CRISPR/Cas9技术在这些感染性疾病中应用的研究进展。
基金financially supported by the National Key Research and Development Program of China(No.2017YFD0500103)the Beijing Natural Science Foundation(No.5152023)+4 种基金the National Natural Science Foundation of China(No.31772747 and31272385)the Jilin Province Science and Technology Development Projects(20150204077NY)the Graduate Innovation Fund of Jilin Universitythe Program for Chang jiang Scholarsthe University Innovative Research Team(No.IRT1248)
文摘CRISPR/Cas9-mediated genome editing can inhibit virus infection by targeting the conserved regions of the viral genomic DNA. Unexpectedly, we found previously that pseudorabies virus(PRV) could escape from CRISPR/Cas9-mediated inhibition.In order to elucidate whether the escape of PRV from Cas9-mediated inhibition was due to cell deficiencies, such as genetic instability of sgRNA or Cas9 protein, the positive cells were passaged ten times, and PRV infection in the sgRNA-expressing cells was evaluated in the present study. The results showed that subculturing cells has no effect on Cas9-mediated cleavage of PRV. Different passages of PX459-PRV cells can stably express sgRNA to facilitate Cas9/sgRNA cleavage on the UL30 gene of PRV, resulting in a pronounced inhibition of PRV infection. Studies to elucidate the mechanism of PRV escape are currently in progress.
文摘BACKGROUND： Visualization of chromosomal loci location and dynamics is crucial for understanding many fundamental intra-nuclear processes such as DNA transcription, replication, and repair. OBJECTIVE： Here, we will describe the development of fluorescence labeling methods for chromatin imaging, including traditional as well as emerging chromatin labeling techniques in both fixed and live cells. We will also discuss current issues and provide a perspective on future developments and applications of the chromatin labeling technology. METHODS： A systematic literature search was performed using the PubMed. Studies published over the past 50 years were considered for review. More than 100 articles were cited in this review. RESULTS： Taking into account sensitivity, specificity, and spatiotemporal resolution, fluorescence labeling and imaging has been the most prevalent approach for chromatin visualization. Among all the fluorescent labeling tools, the adoption ofgenome editing tools, such as TALE and CRISPR, have great potential for the labeling and imaging of chromatin. CONCLUSION： Although a number of chromatin labeling techniques are available for both fixed and live cells, much more effort is still clearly required to develop fluorescence labeling methods capable of targeting arbitrary sequences non-intrusively to allow long-term, multiplexing, and high-throughput imaging of genomic loci and chromatin structures. The emerging technological advances will outline a next-generation effort toward the comprehensive delineation of chromatin at single-cell level with single-molecule resolution.
