Files and links (1)
Berman_Rachel_202648.67 MB
PDF Embargoed Access, Embargo ends: 31 May 2028
Dissertation
Defining novel Cas:gRNA pairings most effective at targeting the integrated HIV-1 genome
Doctor of Philosophy (Ph.D.), Drexel University
May 2026
DOI:
https://doi.org/10.17918/00011360
Abstract
The dynamic latent proviral reservoir remains the principal barrier to a broad-spectrum cure for human immunodeficiency virus type 1 (HIV-1). Once an acute threat, HIV-1 is now a chronic disease for people living with HIV-1 (PLWH) who adhere to antiretroviral therapy (ART), though the latent reservoir endures, contributing to chronic disease syndromes, neuroinflammation, and HIV-1-associated neurocognitive disorders (HAND). Curative strategies for HIV-1 must target viral reservoirs across compartmental barriers, while navigating extensive inter- and intra-patient quasispecies diversity without causing additional harm to those undergoing treatment. Clustered Regularly Interspersed Short Palindromic repeats (CRISPR) gene editing has been proposed as a curative strategy, and the Staphylococcus aureus Cas9 (SaCas9) ortholog has emerged as the leading endonuclease for in vivo cure therapies. However, the two main components of CRISPR - the endonuclease and the guide RNA (gRNA) - remain poorly characterized in a comprehensive, Cas-ortholog and HIV-1-specific manner. Herein, the Cas:gRNA pairing for an HIV-1 cure strategy are evaluated computationally and experimentally. Computational interrogation of protospacer adjacent motif (PAM) availability identified the potential for safe, broad and effective (SBE) gRNA yield across HIV-1 quasispecies through the Nominate, Diversify, Narrow and Filter (NDNF) pipeline and characterized the trade-off between PAM breadth and off-target potential across a wide range of natural and engineered Cas orthologs. Experimentally, the optimal SaCas9:gRNA pair was interrogated through delivery of a 6,000 HIV-1-specific gRNA library to J-Lat 10.6 T lymphocytes followed by PMA/ionomycin stimulation, fluorescence-activated cell sorting (FACS), and next generation sequencing. Weighted comparisons between time points, reactivated, and silenced cell populations produced a ranked annotation of all 6,000 protospacers. Top SaCas9 gRNAs were distributed across multiple loci throughout the proviral genome, with the leading gRNA targeting vif. Critically, this analysis revealed a nuanced positional and nucleotide-specific mismatch preference for SaCas9 gRNAs that diverges from current design frameworks. Site-specific sequence logos derived from this analysis provide a practical framework for optimal SaCas9 gRNA design. Together these findings characterize the targeted components of CRISPR for an HIV-1 cure strategy and represent a significant advance toward a durable, safe and universal cure for HIV-1.
Metrics
1 Record Views
Details
- Title
- Defining novel Cas:gRNA pairings most effective at targeting the integrated HIV-1 genome
- Creators
- Rachel Elizabeth Berman
- Contributors
- Brian Wigdahl (Advisor)Michael R. Nonnemacher (Advisor)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University
- Number of pages
- xxii, 462 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Biochemistry and Molecular Biology; College of Arts and Sciences; Drexel University
- Other Identifier
- 991022181872804721