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Dissertation
Design and validation of broad-spectrum guide RNAs for CRISPR/Cas9-mediated HIV-1 elimination and cure
Doctor of Philosophy (Ph.D.), Drexel University
May 2018
DOI:
https://doi.org/10.17918/D8066R
Abstract
The CRISPR/Cas9 gene-editing system is effective at disrupting and excising the entire HIV-1 proviral genome from infected cells by targeting the long terminal repeats (LTRs). Of the currently available gRNAs, we have shown that only a few have any capability to target patient-derived HIV-1 quasispecies (vQS). Consequently, there is a pressing need to identify gRNAs to target all vQS in large numbers of patients. This thesis focuses on pioneering advances that we have made in designing/testing broad-spectrum gRNAs targeting the HIV-1 LTR. Studies utilizing 48 patient-derived samples, determined that a hybrid method of one personalized and 3 broad-spectrum gRNAs can target 45 of 48 patient-derived sequences from the Drexel CNS AIDS Research and Eradication Study (CARES) cohort. Given this, it was hypothesized that a novel set of broad-spectrum gRNAs could be designed by increasing the number of patient sequences. Therefore, LTRs from 269 patients were deep-sequenced and a novel package of select gRNAs were analyzed. These gRNAs were predicted to cleave all patient-derived sequences in the CARES cohort and all subtype B sequences in the Los Alamos National Laboratory HIV database without predicted off-target effects. Numerous functional assays demonstrated that these gRNAs were more efficacious than those previously published gRNAs, especially at cleaving patient-derived HIV-1 sequences. In addition, the selected gRNAs were highly effective at targeting other HIV subtypes, thus further demonstrating their broad-spectrum capabilities. Overall, the newly designed gRNAs target the vQS spectrum in large numbers of HIV-1-infected patients and bring us closer to a potential HIV cure.
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Details
- Title
- Design and validation of broad-spectrum guide RNAs for CRISPR/Cas9-mediated HIV-1 elimination and cure
- Creators
- Neil Thomas Sullivan - DU
- Contributors
- Brian Wigdahl (Advisor) - Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- various pagings
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Microbiology and Immunology; College of Medicine; Drexel University
- Other Identifier
- 7961; 991014632414804721