Illuminating single genomic loci in live cells by reducing nuclear background fluorescence

作     者：Song Lu Dianbing Wang Yu Hou Dongge Guo Yulin Deng Xian-En Zhang Song Lu;Dianbing Wang;Yu Hou;Dongge Guo;Yulin Deng;Xian-En Zhang

作者机构：School of Life SciencesBeijing Institute of TechnologyBeijing 100081China National Laboratory of BiomacromoleculesCAS Center for Excellence in BiomacromoleculesInstitute of BiophysicsChinese Academy of SciencesBeijing 100101China University of Chinese Academy of SciencesBeijing 100049China 

出 版 物：《Science China(Life Sciences)》 (中国科学（生命科学英文版）)

年 卷 期：2021年第64卷第5期

页      面：667-677页

核心收录：

学科分类：0710[理学-生物学] 0711[理学-系统科学] 0830[工学-环境科学与工程（可授工学、理学、农学学位）] 07[理学] 

基　　金：the National Natural Science Foundation of China(21890743) Strategic Priority Research Program of the Chinese Academy of Sciences,China(XDB29050100) National Key Research and Development Program of China(2017YFA0205503)。 

主　　题：CRISPR/dCas9 labeling background reduction low-repetitive loci live cell imaging 

摘      要：The tagging of genomic loci in living cells provides visual evidence for the study of genomic spatial organization and gene interaction.CRISPR/dCas9(clustered regularly interspaced short palindromic repeats/deactivated Cas9)labeling system labels genes through binding of the dCas9/sgRNA/fluorescent protein complex to repeat sequences in the target genomic loci.However,the existence of numerous fluorescent proteins in the nucleus usually causes a high background fluorescent readout.This study aims to limit the number of fluorescent modules entering the nucleus by redesigning the current CRISPR/dCas9-SunTag labeling system consisting of dCas9-SunTag-NLS(target module)and scFv-sfGFP-NLS(signal module).We removed the nuclear location sequence(NLS)of the signal module and inserted two copies of EGFP into the signal module.The ratio of the fluorescent intensity of the nucleus to that of the cytoplasm(N/C ratio)was decreased by 71%,and the ratio of the signal to the background(S/B ratio)was increased by 1.6 times.The system can stably label randomly selected genomic loci with as few as 9 repeat sequences.