Two-Color Imaging of Nonrepetitive Endogenous Loci in Human Cells

Ipek Tasan, Christina Jean Su, Behnam Enghiad, Meng Zhang, Shekhar Mishra, Huimin Zhao

Research output: Contribution to journalArticlepeer-review

Abstract

Tools for live cell imaging of multiple nonrepetitive genomic loci in mammalian cells are necessary to study chromatin dynamics. Here, we report a new system based on two chromosomally integrated orthogonal irregular repeat arrays and particularly a new general strategy to construct irregular repeat arrays. Briefly, we utilized a "bridge oligonucleotide-mediated ligation"protocol to assemble 8-mer repeats de novo which were then combined into a final 96-mer repeat array using Golden Gate cloning. This strategy was used for assembling a new mutant TetO irregular repeat array, which worked orthogonally to the wild type TetO repeat. Single copy integration of the new repeat array did not cause replication deficiencies at the tagged locus. Moreover, the mutant TetO irregular repeat could also be visualized by CRISPR imaging. Our new irregular repeat assembly method demonstrates a generally applicable strategy that can be used for assembling additional orthogonal repeat arrays for imaging genomic loci and irregular repeats to visualize RNA or proteins via signal amplification.

Original languageEnglish (US)
Pages (from-to)2502-2514
Number of pages13
JournalACS synthetic biology
Volume9
Issue number9
DOIs
StatePublished - Sep 18 2020

Keywords

  • CRISPR/Cas9
  • FROS (fluorescent repressor operator system)
  • fluorescence microscopy
  • genome editing
  • irregular repeat array assembly
  • live cell imaging

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

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