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
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.
