Live‐cell imaging of genomic loci with Cas9 variants

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