He Xiaohui, Sun Yadong, Ma Hanhui
School of Life Science and Technology, ShanghaiTech University, Shanghai, China.
PLoS Biol. 2025 Jan 24;23(1):e3003009. doi: 10.1371/journal.pbio.3003009. eCollection 2025 Jan.
The organization of the human genome in space and time is critical for transcriptional regulation and cell fate determination. However, robust methods for tracking genome organization or genomic interactions over time in living cells are lacking. Here, we developed a multicolor DNA labeling system, ParSite, to simultaneously track triple genomic loci in the U2OS cells. The tricolor ParSite system is derived from the T. thermophilus ParB/ParSc (TtParB/ParSc) system by rational design. We mutated the interface between TtParB and ParSc and generated a new pair of TtParBm and ParSm for genomic DNA labeling. The insertions of 16 base-pair palindromic ParSc and ParSm into genomic loci allow dual-color DNA imaging in living cells. A pair of genomic loci labeled by ParSite could be colocalized with p53-binding protein 1 (53BP1) in response to CRISPR/Cas9-mediated double-strand breaks (DSBs). The ParSite permits tracking promoter and terminator dynamics of the APP gene, which spans 290 kilobases in length. Intriguingly, the hybrid ParS (ParSh) of half-ParSc and half-ParSm enables for the visualization of a third locus independent of ParSc or ParSm. We simultaneously labeled 3 loci with a genomic distance of 36, 89, and 352 kilobases downstream the C3 repeat locus, respectively. In sum, the ParSite is a robust DNA labeling system for tracking multiple genomic loci in space and time in living cells.
人类基因组在空间和时间上的组织对于转录调控和细胞命运决定至关重要。然而，目前缺乏在活细胞中随时间追踪基因组组织或基因组相互作用的强大方法。在这里，我们开发了一种多色DNA标记系统ParSite，用于同时追踪U2OS细胞中的三个基因组位点。三色ParSite系统是通过合理设计从嗜热栖热菌ParB/ParSc（TtParB/ParSc）系统衍生而来的。我们对TtParB和ParSc之间的界面进行了突变，并生成了一对新的TtParBm和ParSm用于基因组DNA标记。将16个碱基对的回文ParSc和ParSm插入基因组位点可实现活细胞中的双色DNA成像。响应CRISPR/Cas9介导的双链断裂（DSB），由ParSite标记的一对基因组位点可与p53结合蛋白1（53BP1）共定位。ParSite允许追踪APP基因的启动子和终止子动态，该基因跨度长达290千碱基。有趣的是，半ParSc和半ParSm的杂交ParS（ParSh）能够可视化独立于ParSc或ParSm的第三个位点。我们同时标记了C3重复位点下游基因组距离分别为36、89和352千碱基的3个位点。总之，ParSite是一种强大的DNA标记系统，用于在活细胞中随时间追踪多个基因组位点。
