Dissertation
Identification and evaluation of guide RNA design determinants for anti-HIV-1 therapy using CRISPR/Cas9
Doctor of Philosophy (Ph.D.), Drexel University
Feb 2020
DOI:
https://doi.org/10.17918/00001026
Abstract
Genome editing of latently integrated HIV-1 provirus using CRISPR/Cas9 has shown promising therapeutic potential to deliver a cure for the disease. The RNA-guided gene editing system is comprised of a guide RNA (gRNA) and Cas9 protein that induces genomic edits at targeted regions defined by the complementary sequence in the gRNA. Previous results using CRISPR/Cas9 to inactivate HIV-1 replication have achieved long-term HIV-1 suppression in both in vitro and in vivo infection models. However, there has not been a systematic examination of HIV-1 inactivation efficiency and off-target effects. A comprehensive meta-analysis of all current literature was conducted and important determinants that affect therapeutic outcome have been proposed. This analysis and other growing evidence have shown that the existing HIV-1 sequence variants derived from the latent HIV-1 reservoir reduced therapeutic efficacy and allowed for viral escape. We hypothesized that gRNA design methods considering genetic variation of HIV-1 sequences that are present among infected individuals will enhance efficacy of CRISPR-mediated antiviral therapy, prevent resistance, and decrease off-target effects. These methods led to development of a gRNA targeting HIV-1-specific NF-[kappa]B binding sites in the HIV promoter or long terminal repeat (LTR) that showed moderate efficiency at suppressing HIV-1 replication and low off-target effects. These results have indicated the utility of these prediction tools in future design pipelines. The review of gRNA literature also revealed that there is a lack of research in developing novel tools that target the global HIV-1 strains. Given this observation, a computational tool was developed to produce a list of gRNAs that have been predicted to be effective across global HIV-1 subtypes for further evaluation. In addition, throughout this work, analysis of CRISPR-mediated off-target profiles unraveled the effect of DNA accessibility on CRISPR editing efficiency. The results showed that correlations between sequence similarity and CRISPR-induced cleavage frequency was altered by the presence of cellular factors that modulated the level of DNA accessibility. This dissertation has demonstrated that understanding the genetic variation of HIV-1 and DNA accessibility of the proviral genome is indispensable to a CRISPR-mediated antiviral strategy.
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Details
- Title
- Identification and evaluation of guide RNA design determinants for anti-HIV-1 therapy using CRISPR/Cas9
- Creators
- Cheng-Han Chung
- Contributors
- Brian Wigdahl (Advisor)William Nathanial Dampier (Advisor)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xxi, 271 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Microbiology and Immunology; College of Medicine; Drexel University
- Other Identifier
- 991014695241004721