Date of Completion

Spring 4-27-2018

Project Advisor(s)

Pramod K. Srivatava; Johann P. Gogarten; David Daggett

University Scholar Major

Molecular and Cell Biology

Disciplines

Genetics | Immunology of Infectious Disease | Molecular Genetics | Translational Medical Research

Abstract

The new gene editing system CRISPR/Cas9, composed of a complex composed of a guide RNA and the Cas9 endonuclease, promises to revolutionize biological research and potentially allow clinicians to directly modify patient DNA in vivo. While its applications in the treatment of genetic diseases and in modifying immune cells for immunotherapy are currently being explored, CRISPR/Cas9’s potential utility as a modular system for targeting tumor-specific mutated sequences has not as of yet been explored. While CRISPR/Cas9 is specific enough to target small insertions and deletions or gross chromosomal rearrangements, it is not specific enough to reliably restrict editing to single nucleotide variants (SNVs), which compose the majority of cancer-associated mutations. By searching for tumor-specific SNVs that generate new protospacer adjacent motifs (neoPAMs), a short sequence that must be present next to the target sequence in order for Cas9 to cleave at the target site, gene editing can theoretically be restricted to tumor cells bearing the mutation of interest. This capability could permit the insertion of suicide genes, pro-inflammatory cytokines, or immunogenic epitopes in a tumor-specific manner. The results shown here demonstrate the importance of taking into account guide RNA efficiency when selecting a target site. New target sites in the tumor cell line FABF with high predicted guide RNA efficiencies are identified and discussed. Finally, strategies for maximizing the antitumor effect of neoPAM-restricted gene editing are described and compared.

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