Genome-wide Cas9 binding specificity in Saccharomyces cerevisiae

Zachary J. Waldrip, Piroon Jenjaroenpun, Oktawia DeYoung, Intawat Nookaew, Sean D. Taverna, Kevin D. Raney, Alan J. Tackett

Research output: Contribution to journalArticlepeer-review

Abstract

The CRISPR system has become heavily utilized in biomedical research as a tool for genomic editing as well as for site-specific chromosomal localization of specific proteins. For example, we developed a CRISPR-based methodology for enriching a specific genomic locus of interest for proteomic analysis in Saccharomyces cerevisiae, which utilized a guide RNA-targeted, catalytically dead Cas9 (dCas9) as an affinity reagent. To more comprehensively evaluate the genomic specificity of using dCas9 as a site-specific tool for chromosomal studies, we performed dCas9-mediated locus enrichment followed by next-generation sequencing on a genome-wide scale. As a test locus, we used the ARS305 origin of replication on chromosome III in S. cerevisiae. We found that enrichment of this site is highly specific, with virtually no off-target enrichment of unique genomic sequences. The high specificity of genomic localization and enrichment suggests that dCas9-mediated technologies have promising potential for site-specific chromosomal studies in organisms with relatively small genomes such as yeasts.

Original languageEnglish (US)
Article number9442
JournalPeerJ
Volume8
DOIs
StatePublished - 2020

Keywords

  • CRISPR
  • Cas9
  • ChIP-seq
  • Chromatin Immunoprecipitation
  • Off-target binding

ASJC Scopus subject areas

  • Neuroscience(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

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