Genome-wide Cas9 binding specificity in Saccharomyces cerevisiae

PeerJ

article

Zachary J. Waldrip¹  Piroon Jenjaroenpun²  Oktawia DeYoung¹  Intawat Nookaew²  Sean D. Taverna³  Kevin D. Raney¹  Alan J. Tackett¹ 

[1] Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences;Department of Biomedical Informatics, University of Arkansas for Medical Sciences;Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine

关键词: CRISPR;    ChIP-seq;    Chromatin Immunoprecipitation;    Cas9;    Off-target binding;   

DOI  :  10.7717/peerj.9442

学科分类：社会科学、人文和艺术（综合）

来源: Inra

【 摘 要 】

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.

【 授权许可】

CC BY