CRISPR FISHer enables high-sensitivity imaging of nonrepetitive DNA in living cells through phase separation-mediated signal amplification  

作　　者：Xin-Yuan Lyu Yuan Deng Xiao-Yan Huang Zhen-Zhen Li Guo-Qing Fang Dong Yang Feng-Liu Wang Wang Kang En-Zhi Shen Chun-Qing Song 

机构地区：[1]Research Center for Industries of the Future,School of Life Sciences,Westlake University,Hangzhou,Zhejiang,China [2]Key Laboratory of Growth Regulation and Translational Research of Zhejiang Province,School of Life Sciences,Westlake University,Hangzhou,Zhejiang,China [3]Westlake Laboratory of Life Sciences and Biomedicine,Hangzhou,Zhejiang,China [4]Institute of Basic Medical Sciences,Westlake Institute for Advanced Study,Hangzhou,Zhejiang,China

出　　处：《Cell Research》2022年第11期969-981,共13页细胞研究（英文版）

基　　金：support from the Research Center for Industries of the Future(RCIF)at Westlake University,the Westlake Education Foundation,the National Natural Science Foundation of China(92068103);Westlake Laboratory of Life Sciences.E.-Z.S.acknowledges support from the Westlake Education Foundation and the National Natural Science Foundation of China(32070628).

摘　　要：The dynamic three-dimensional structures of chromatin and extrachromosomal DNA molecules regulate fundamental cellular processes and beyond.However,the visualization of specific DNA sequences in live cells,especially nonrepetitive sequences accounting for most of the genome,is still vastly challenging.Here,we introduce a robust CRISPR-mediated fluorescence in situ hybridization amplifier(CRISPR FISHer)system,which exploits engineered sgRNA and protein trimerization domain-mediated,phase separation-based exponential assembly of fluorescent proteins in the CRISPR-targeting locus,conferring enhancements in both local brightness and signal-to-background ratio and thus achieving single sgRNA-directed visualization of native nonrepetitive DNA loci in live cells.In one application,by labeling and tracking the broken ends of chromosomal fragments,CRISPR FISHer enables real-time visualization of the entire process of chromosome breakage,separation,and subsequent intra-or inter-chromosomal ends rejoining in a single live cell.Furthermore,CRISPR FISHer allows the movement of small extrachromosomal circular DNAs(eccDNAs)and invading DNAs to be recorded,revealing substantial differences in dynamic behaviors between chromosomal and extrachromosomal loci.With the potential to track any specified self or non-self DNA sequences,CRISPR FISHer dramatically broadens the scope of live-cell imaging in biological events and for biomedical diagnoses.

关 键 词：SEPARATION SEQUENCES phase 

分 类 号：Q78[生物学—分子生物学]