Research Mentor(s)

Pollard, Dan A.

Description

A variety of papers published in the last decade have suggested that chromatin accessibility could have significant influence over the success of CRISPR-Cas9 experiments in S. cerevisiae; where chromatin accessibility refers to nucleosome presence at a targeted genomic location. It has been found that nucleosome presence can physically impede Cas9 from making a double stranded break at a specific target site, causing a decreased experimental efficiency. We have created a web-tool called NucJuke that seeks to mitigate this problem by categorically re-ranking gRNAs based on having high, partial, or low chromatin accessibility (referring to no nucleosome occupancy, partial nucleosome occupancy, or full nucleosome occupancy respectively). The intention is for NucJuke to be used as a step in the process of designing CRISPR-Cas9 experiments. This tool utilizes two, single base pair resolution nucleosome maps produced using a chemical approach. This approach utilizes a unique cysteine that can covalently attach to a sulphhydryl-reactive, copper-chelating label. When these are bound, the label will orient in close proximity to the DNA backbone and directly flanking the nucleosome center. This cysteine is introduced to histone H4 in S. cerevisiae and label is subsequently introduced. This results in the formation of short-lived hydroxyl radicals that react with and cleave the DNA backbone at sites flanking the nucleosome center with high specificity, allowing for single base pair resolution of nucleosome centers in yeast. We are currently evaluating genome-wide statistics for how impactful NucJuke could potentially be for CRISPR experiments in yeast. We are also beginning the process of testing the predictions of relative CRISPR efficiency that NucJuke makes to validate and demonstrate the utility of NucJuke.

Document Type

Event

Start Date

15-5-2019 9:00 AM

End Date

15-5-2019 5:00 PM

Location

Carver Gym (Bellingham, Wash.)

Department

Biology

Genre/Form

student projects, posters

Subjects – Topical (LCSH)

Saccharomyces cerevisiae--Biotechnology; CRISPR-associated protein 9; Gene regulatory networks; Chromatin; Computer-aided design

Type

Image

Keywords

CRISPR, Cas9, CRISPR-Cas9, Nucleosome, Yeast, Gene Editing, Webtool, Targeted Mutagenesis, DNA accessibility, Genomics

Rights

Copying of this document in whole or in part is allowable only for scholarly purposes. It is understood, however, that any copying or publication of this document for commercial purposes, or for financial gain, shall not be allowed without the author’s written permission.

Language

English

Format

application/pdf

Share

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May 15th, 9:00 AM May 15th, 5:00 PM

NucJuke: A web tool for re-ranking CRISPR-Cas9 gRNAs based on chromatin accessibility in yeast

Carver Gym (Bellingham, Wash.)

A variety of papers published in the last decade have suggested that chromatin accessibility could have significant influence over the success of CRISPR-Cas9 experiments in S. cerevisiae; where chromatin accessibility refers to nucleosome presence at a targeted genomic location. It has been found that nucleosome presence can physically impede Cas9 from making a double stranded break at a specific target site, causing a decreased experimental efficiency. We have created a web-tool called NucJuke that seeks to mitigate this problem by categorically re-ranking gRNAs based on having high, partial, or low chromatin accessibility (referring to no nucleosome occupancy, partial nucleosome occupancy, or full nucleosome occupancy respectively). The intention is for NucJuke to be used as a step in the process of designing CRISPR-Cas9 experiments. This tool utilizes two, single base pair resolution nucleosome maps produced using a chemical approach. This approach utilizes a unique cysteine that can covalently attach to a sulphhydryl-reactive, copper-chelating label. When these are bound, the label will orient in close proximity to the DNA backbone and directly flanking the nucleosome center. This cysteine is introduced to histone H4 in S. cerevisiae and label is subsequently introduced. This results in the formation of short-lived hydroxyl radicals that react with and cleave the DNA backbone at sites flanking the nucleosome center with high specificity, allowing for single base pair resolution of nucleosome centers in yeast. We are currently evaluating genome-wide statistics for how impactful NucJuke could potentially be for CRISPR experiments in yeast. We are also beginning the process of testing the predictions of relative CRISPR efficiency that NucJuke makes to validate and demonstrate the utility of NucJuke.

 

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