Live-cell imaging of genomic loci with Cas9 variants

Biotechnol J. 2022 Dec;17(12):e2100381. doi: 10.1002/biot.202100381. Epub 2022 Sep 4.

Abstract

Background: Endonuclease-deactivated clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (dCas9) has been repurposed for live-cell imaging of genomic loci. Engineered or evolved dCas9 variants have been developed to increase the applicability of the CRISPR/dCas9 system. However, there have been no systematic comparisons of these dCas9 variants in terms of their performance in the visualization of genomic loci.

Main methods and major results: Here we demonstrate that dSpCas9 and its variants deSpCas9(1.1), dSpCas9-HF1, devoCas9, and dxCas9(3.7) can be used for CRISPR-based live-cell genomic imaging. dSpCas9 had the greatest utility, with a high labeling efficiency of repetitive sequences-including those with a low number of repeats-and good compatibility with target RNA sequences at the MUC4 locus that varied in length from 13 to 23 nucleotides. We combined CRISPR-Tag with the dSpCas9 imaging system to observe the dynamics of the Tet promoter and found that its movement was restricted when it was active.

Conclusions and implications: These novel Cas9 variants provide a new set of tools for investigating the spatiotemporal regulation of gene expression through live imaging of genomic sites.

Keywords: CRISPR/Cas9; dCas9 variants; dynamic changes; live-cell imaging.

MeSH terms

  • CRISPR-Cas Systems* / genetics
  • Endonucleases* / genetics
  • Genetic Loci
  • Genomics
  • RNA

Substances

  • Endonucleases
  • RNA