Presenter: Sahar Kanishka, College of Science, School of Biological Sciences
Authors: Sahar N. Kanishka, Evan P. Drage, James A. Gagnon
Faculty Advisor: James Gagnon, College of Science, School of Biological Sciences
Institution: University of Utah
Lineage tracing is an essential tool for understanding developmental biology. The ability to trace single cells back to their descendants has allowed for rapid advancements in understanding the mechanisms of development. New lineage tracing tools allow for more efficient reconstruction of lineage trees. One new approach for lineage tracing uses the CRISPR-Cas9 system to generate a multitude of edits within the genome, known as barcodes, and then relates single cells to one another based on shared mutations. However, there are three key limitations of the CRISPR-Cas9 system for lineage tracing. One limitation is the inability to induce specific mutations; instead, CRISPR-Cas9 generates a range of random edits by initiating a double-stranded break (DSB). CRISPR lineage tracing would be more accurate and predictable if we could generate sets of user-defined mutations. A second limitation is that DSBs can delete large regions of the genome, or can lead to the death of the cell. The third limitation is the inability to visualize the spatial context of barcoded cells. Since the barcodes are composed of random edits, they are not compatible with standard imaging methods. CRISPR prime editing is a new genome editing tool that has the potential to overcome these limitations. Prime editing uses a modified sgRNA known as a pegRNA and Cas9 fused to a reverse transcriptase enzyme; these components work together to “write” specific sequences into the genome. I hypothesize that prime editing has the potential to overcome limitations of CRISPR lineage tracing. I will first optimize prime editing in zebrafish. Then I will utilize the prime editing system for lineage tracing by inserting specific sequences. Finally, I will use in situ hybridization to recover the spatial context of barcoded cells. This project will develop a new strategy for lineage tracing using prime editing in zebrafish.