CRISPR-Cas-Mediated Tethering Recruits the Yeast HMR Mating-Type Locus to the Nuclear Periphery but Fails to Silence Gene Expression

ACS Synth Biol. 2021 Nov 19;10(11):2870-2877. doi: 10.1021/acssynbio.1c00306. Epub 2021 Nov 1.

Abstract

To investigate the relationship between genome structure and function, we have developed a programmable CRISPR-Cas system for nuclear peripheral recruitment in yeast. We benchmarked this system at the HMR and GAL2 loci, both of which are well-characterized model systems for localization to the nuclear periphery. Using microscopy and gene silencing assays, we demonstrate that CRISPR-Cas-mediated tethering can recruit the HMR locus but does not detectably silence reporter gene expression. A previously reported Gal4-mediated tethering system does silence gene expression, and we demonstrate that the silencing effect has an unexpected dependence on the properties of the protein tether. The CRISPR-Cas system was unable to recruit GAL2 to the nuclear periphery. Our results reveal potential challenges for synthetic genome structure perturbations and suggest that distinct functional effects can arise from subtle structural differences in how genes are recruited to the periphery.

Keywords: CRISPR-Cas; HMR; dCas9; gene silencing; genome structure; nuclear periphery.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems / genetics*
  • Cell Nucleus / genetics*
  • DNA-Binding Proteins / genetics
  • Gene Expression / genetics*
  • Gene Silencing / physiology*
  • Genes, Reporter / genetics
  • Genetic Techniques
  • Genome, Bacterial / genetics
  • Saccharomyces cerevisiae / genetics*

Substances

  • DNA-Binding Proteins