title: Long-term dual-color tracking of genomic loci by modified sgRNAs of the CRISPR/Cas9 system.
abstract: Visualization of chromosomal dynamics is important for understanding many fundamental intra-nuclear processes. Efficient and reliable live-cell multicolor labeling of chromosomal loci can realize this goal. However, the current methods are constrained mainly by insufficient labeling throughput, efficiency, flexibility as well as photostability. Here we have developed a new approach to realize dual-color chromosomal loci imaging based on a modified single-guide RNA (sgRNA) of the CRISPR/Cas9 system. The modification of sgRNA was optimized by structure-guided engineering of the original sgRNA, consisting of RNA aptamer insertions that bind fluorescent protein-tagged effectors. By labeling and tracking telomeres, centromeres and genomic loci, we demonstrate that the new approach is easy to implement and enables robust dual-color imaging of genomic elements. Importantly, our data also indicate that the fast exchange rate of RNA aptamer binding effectors makes our sgRNA-based labeling method much more tolerant to photobleaching than the Cas9-based labeling method. This is crucial for continuous, long-term tracking of chromosomal dynamics. Lastly, as our method is complementary to other live-cell genomic labeling systems, it is therefore possible to combine them into a plentiful palette for the study of native chromatin organization and genome ultrastructure dynamics in living cells.
authors: State Key Laboratory of Membrane Biology, Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing 100871, China.
authors: Shipeng
authors: S
authors: Shao
authors: State Key Laboratory of Membrane Biology, Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing 100871, China.
authors: Weiwei
authors: W
authors: Zhang
authors: Center for Quantitative Biology, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China.
authors: Huan
authors: H
authors: Hu
authors: State Key Laboratory of Membrane Biology, Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing 100871, China.
authors: Boxin
authors: B
authors: Xue
authors: State Key Laboratory of Membrane Biology, Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing 100871, China.
authors: Jinshan
authors: J
authors: Qin
authors: State Key Laboratory of Membrane Biology, Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing 100871, China.
authors: Chaoying
authors: C
authors: Sun
authors: State Key Laboratory of Membrane Biology, Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing 100871, China.
authors: Yuao
authors: Y
authors: Sun
authors: Biodynamic Optical Imaging Center (BIOPIC), Peking-Tsinghua Center for Life Sciences, School of Life Sciences, Peking University, Beijing 100871, China.
authors: Wensheng
authors: W
authors: Wei
authors: State Key Laboratory of Membrane Biology, Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing 100871, China sun_yujie@pku.edu.cn.
authors: Yujie
authors: Y
authors: Sun
mesh_terms: D052157:Aptamers, Nucleotide / Q000235:genetics; D064113:CRISPR-Cas Systems / Q000235:genetics*; D045744:Cell Line, Tumor; D002503:Centromere / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D005456:Fluorescent Dyes / Q000737:chemistry; D056426:Genetic Loci / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D013194:Staining and Labeling / Q000379:methods*; D016615:Telomere / Q000378:metabolism
chemical_list: D052157:Aptamers, Nucleotide; D002843:Chromatin; D005456:Fluorescent Dyes; D000094704:RNA, Guide, CRISPR-Cas Systems