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

doi:10.7717/peerj.9442

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

School of Medicine

Research output: Contribution to journal › Article › peer-review

1 Scopus citations

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 language	English (US)
Article number	9442
Journal	PeerJ
Volume	8
DOIs	https://doi.org/10.7717/peerj.9442
State	Published - 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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title = "Genome-wide Cas9 binding specificity in Saccharomyces cerevisiae",

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.",

keywords = "CRISPR, Cas9, ChIP-seq, Chromatin Immunoprecipitation, Off-target binding",

author = "Waldrip, {Zachary J.} and Piroon Jenjaroenpun and Oktawia DeYoung and Intawat Nookaew and Taverna, {Sean D.} and Raney, {Kevin D.} and Tackett, {Alan J.}",

note = "Funding Information: The following grants from the United States National Institutes of Health (NIH) provided funding for this project: National Institute of General Medical Sciences (NIGMS) R35GM122601 awarded to Kevin D. Raney, NIGMS R01GM118760 awarded to Sean D. Taverna, NIGMS P20GM121293 and P20GM103429 awarded to Alan J. Tackett, and National Institute on Drug Abuse (NIDA) R21DA041822 awarded to Alan J. Tackett. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Publisher Copyright: {\textcopyright} Copyright 2020 Waldrip et al.",

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language = "English (US)",

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journal = "PeerJ",

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AU - Waldrip, Zachary J.

AU - Jenjaroenpun, Piroon

AU - DeYoung, Oktawia

AU - Nookaew, Intawat

AU - Taverna, Sean D.

AU - Raney, Kevin D.

AU - Tackett, Alan J.

N1 - Funding Information: The following grants from the United States National Institutes of Health (NIH) provided funding for this project: National Institute of General Medical Sciences (NIGMS) R35GM122601 awarded to Kevin D. Raney, NIGMS R01GM118760 awarded to Sean D. Taverna, NIGMS P20GM121293 and P20GM103429 awarded to Alan J. Tackett, and National Institute on Drug Abuse (NIDA) R21DA041822 awarded to Alan J. Tackett. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Publisher Copyright: © Copyright 2020 Waldrip et al.

PY - 2020

Y1 - 2020

N2 - 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.

AB - 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.

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Genome-wide Cas9 binding specificity in Saccharomyces cerevisiae

Abstract

Keywords

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