Optimizing an AAV-CRISPR system for retinal gene therapy

Thesis

Introduction: CRISPR/Cas9 is a bacterial defence system which evolved to cleave and deactivate bacteriophage DNA. The elements involved in the CRISPR/Cas9 system may be modified for gene therapy. CRISPR/Cas9 consists of two components, a single guide RNA (sgRNA) and a Cas9 protein. The sgRNA allows for sequence-specific binding. “Active” Cas9 creates a double-stranded break in the target region while the “deactivated” form can inhibit transcription. CRISPR/Cas9 can be packaged within adeno-associated viral vectors, which opens the possibility of using CRISPR/Cas9 to target mutations in the retina. We examine several optimizations to the AAV-CRISPR/Cas9 system in vitro in HEK293-eGFP cells.

Materials and Methods: We compare the effects of sgRNA orientation in both active SaCas9 constructs and deactivated SaCas9 constructs with the KRAB repressor (dSaCas9-KRAB). We also examine the cleavage ability of GeoCas9.

Results: There was no difference in Cas9 and sgRNA levels between the in cis and in trans SaCas9 constructs. We also found no difference in Cas9 levels, sgRNA levels, and target knock-down between the cis and trans orientations of the dSaCas9-KRAB constructs. For GeoCas9, we found that DNA cleavage levels were undetectable, despite confirmed expression of both sgRNA and GeoCas9 protein.

Conclusion: When delivering CRISPR/Cas9 in an AAV-based expression cassette, the efficacy appears unrelated to the orientation of the sgRNA (in cis or in trans) relative to the Cas9 gene. Furthermore, GeoCas9 may not be as efficacious as SaCas9, since it was unable to cleave DNA detectably in similar experiments.

Authors: Stevanovic, M

• DOI: 10.5287/ora-8g5dpnpjz
• Type of award: MSc by Research
• Level of award: Masters
• Awarding institution: University of Oxford
• Language: English
• Keywords: CRISPR/Cas9; Gene therapy; Retina; Retinal degeneration
• Subjects: Retinal degeneration; Gene therapy