Imaging Method Using CRISPR/dCas9 and Engineered gRNA Scaffolds Can Perturb Replication Timing at the HSPA1 Locus

Xiong Xiong, Ipek Tasan, Che Yang, Meng Zhang, Gabriela A. Hernandez Gonzalez, Shuting Liu, Pankaj Chaturvedi, Andrew S. Belmont, Huimin Zhao

Research output: Contribution to journalArticlepeer-review

Abstract

Fluorescence microscopy imaging of specific chromosomal sites is essential for genome architecture research. To enable visualization of endogenous loci in mammalian cells, programmable DNA-binding proteins such as TAL effectors and CRISPR/dCas9 are commonly utilized. In addition, site-specific insertion of a TetO repeat array, coupled with TetR-enhanced green fluorescent protein fusion protein expression, can be used for labeling nonrepetitive endogenous loci. Here, we performed a comparison of several live-cell chromosome tagging methods, including their effect on subnuclear positioning, expression of adjacent genes, and DNA replication timing. Our results showed that the CRISPR-based imaging method can delay DNA replication timing and sister chromatid resolution at certain region. However, subnuclear localization of the labeled locus and gene expression from adjacent loci were unaffected by either TetO/TetR or CRISPR-based methods, suggesting that CRISPR-based imaging could be used for applications that do not require DNA replication analysis.

Original languageEnglish (US)
Pages (from-to)1424-1436
Number of pages13
JournalACS synthetic biology
Volume12
Issue number5
DOIs
StatePublished - May 19 2023

Keywords

  • CRISPR-Sirius
  • DNA replication
  • HSPA1 gene
  • TAL effector
  • Tet operator/Tet repressor
  • imaging
  • site-specific targeting

ASJC Scopus subject areas

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

Fingerprint

Dive into the research topics of 'Imaging Method Using CRISPR/dCas9 and Engineered gRNA Scaffolds Can Perturb Replication Timing at the HSPA1 Locus'. Together they form a unique fingerprint.

Cite this