Using Zinc Finger Nucleases for Targeted Genome Modification in the Zebrafish
- Author(s): McCammon, Jasmine Mali
- Advisor(s): Amacher, Sharon L
- et al.
Over the last several decades, the zebrafish has been developed into a powerful laboratory model organism. There is a burgeoning wealth of reverse genetic tools and conditional expression systems available to zebrafish researchers, although some of them have certain weaknesses and others are still not developed to their full potential. Because a reliable embryonic stem cell system has yet to be established, targeted knockouts and more subtle manipulations at endogenous loci have not been achieved in the zebrafish. To address this issue, I turned to zinc finger nucleases (ZFNs), which have been used for such manipulations in other systems. ZFNs are a chimeric fusion between zinc finger proteins (ZFPs), which can be engineered and designed to recognize specific DNA sequences, and the non-specific cleavage domain of the FokI endonuclease. Once the ZFPs bind to the targeted position, FokI will cleave the sequence and create a double strand break (DSB). The cell has two major pathways to repair DSBs: non-homologous end joining (NHEJ) and homology directed repair (HDR). In NHEJ, broken DNA ends are often misaligned, which leads to the incorporation of small insertions and deletions. HDR uses a homologous template to direct repair of the DSB. An exogenously supplied template, with significant regions of homology flanking the DSB, can also be used to direct repair and to "trick" the cell into incorporating novel sequence elements into endogenous loci. Both of these DSB repair pathways confer different ZFN-induced modifications: NHEJ for targeted mutagenesis, and HDR for a variety of manipulations including knock-ins and creation of conditional alleles. I have shown by proof-of-principle that ZFNs can be used efficiently for generating targeted knockouts in somatic cells and in the germline in zebrafish. Because there is a strong bias for DSB repair pathway choice in the zebrafish, ZFN-mediated HDR has proven to be a more challenging task. I have shown evidence of HDR working in the soma, and continue to screen for events in the germline, which is a much smaller population of cells.