Candidates with a keen interest in targeted gene modification and prior hands-on experience with molecular biology and cell culture are preferred. Experience with CRISPR would be an additional advantage. A possibility of Ph.D. enrolment exists, but is not guaranteed.
Work will involve DNAzyme construction, DNAzyme library creation and verification, recombinant protein purification, CRISPR reagent construction including gRNA design, in vitro transcription, and extensive mammalian cell culture, transfection, and screening including analysis of viability and marker expression.
CRISPR-Cas9 technology is a recent advancement in genetic engineering allowing targeted gene disruption with high efficiency. The technology can also be used for targeted insertion of DNA sequences, but less efficiently.
The overall goal of this proposal is to use DNAzymes to facilitate high-efficiency targeted gene insertion. Specific objectives:
- Proof-of-concept studies using a DNAzyme targeting DNA ligase IV, to inhibit NHEJ and promoter HDR mediated gene insertion.
- Construction of the randomized DNAzyme library in a vector suitable for in vivo expression in cell lines.
- Screening and identification of HDR enhancers
- Validation of the top 5 hits from the screen for further characterization and testing.