Screening highly-specific hairpin guide RNAs for CRISPR-Cas gene editing
- UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
- Miranda R. Roesing (Creator)
- Institution
- The University of North Carolina at Greensboro (UNCG )
- Web Site: http://library.uncg.edu/
- Advisor
- Eric Josephs
Abstract: This thesis outlines the development of a screening method to improve the specificity of the CRISPR effector, Cas9, in gene editing applications. We accomplish this by identifying modifications to the RNA-cofactor of Cas9, its guide RNA (gRNA), that determines the DNA sequence recognized by the effector, where changes to the gRNA secondary structure are hypothesized to limit the potential for mutations at unintended sites. The identification of highly specific gRNA sequences can potentially lead to highly specific gene editing techniques that can be used to treat genetic diseases such as Tay-Sachs disease, cystic fibrosis, and sickle cell anemia without off-target mutations. Randomized libraries of gRNAs with extra nucleotides that can form secondary structures such as “hairpins” with the targeting segment of the gRNA (hairpin-gRNAs or hp-gRNAs) are screened with SpyCas9 in the presence of DNA with the intended target, and DNA containing “off-target” sequences that are similar but not an exact match for the target. The results from this screen can provide insights to the biophysical requirements that dictate target recognition and the potential for design rules for high-fidelity hp-gRNAs for any genetic target for use in effective gene therapeutics.
Screening highly-specific hairpin guide RNAs for CRISPR-Cas gene editing
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Created on 5/1/2021
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Additional Information
- Publication
- Thesis
- Language: English
- Date: 2021
- Keywords
- CRISPR-Cas, Gene editing, Specificity, SpyCas9
- Subjects
- CRISPR-associated protein 9
- Gene editing