Líffræðifélag Íslands - biologia.is
Líffræðiráðstefnan 2017
Erindi/veggspjald / Talk/poster V29
Höfundar / Authors: Hörður Guðmundsson, Sigríður Rut Franzdóttir, Zophonías O. Jónsson
Starfsvettvangur / Affiliations: Institute of Life- and Environmental Sciences and Biomedical Center, University of Iceland. Askja, Sturlugata 7, 101 Reykjavik, Iceland.
Kynnir / Presenter: Hörður Guðmundsson
CRISPR is an ancient defense mechanism against viruses in bacteria. The system has been adapted for genome editing in other organisms (CRISPR/Cas9). Briefly, DNA at a specific location defined by a guide RNA (gRNA) is cleaved by the Cas9 nuclease. The method can be used to mutate a gene or insert new DNA via homology repair. Mutated Cas9 can also be used as to activate/repress of gene expression or for visualizing genomic regions.
We have used the CRISPR-Cas9 method to seamlessly tag fly proteins with florescent and epitope markers. Donor plasmids and gRNAs were designed to target both the 5’ and 3’ ends of the coding regions of each gene (for N- and C-terminal fusions). The donor plasmids carried 1kbp sequences homologous to the genomic target region on each side of the marker sequence (GFP, mCherry or FLAG). As part of the process, we set up a system that allows easy design of assembly primers for introducing the same tags into donor plasmids for any gene of interest (using Gibson assembly). Both the donor and gRNA plasmids were injected into fly embryos carrying the Cas9 nuclease and germline transformants were detected by PCR-screening. The efficiency of gRNAs can vary greatly and our yield has been between 4%-30%. The new strains are powerful tools to study the behavior and interactions of native proteins without over-expression, and have been used for visualization of the proteins in vivo, immunoprecipitation and RIP-seq experiments.