Science
104 Lineage Tracing in Zebrafish with CRISPR Prime Editing
Sahar Kanishka and James A. Gagnon (School of Biological Sciences)
Faculty Mentor: James Gagnon (School of Biological Sciences, University of Utah)
All embryos develop from a single cell. We use lineage tracing to map the relationships between individual cells and back to the initial founding cell. These lineage trees can help us understand how cells acquire their fates during normal development, and how that can go wrong in human disease. An emerging method for lineage tracing in embryos uses cellular barcodes. Cellular barcodes individually tag cells with a unique set of mutations specific to that cell. As cell divisions occur, the barcode is passed on to the progeny cells and a lineage tree can constructed based on cells that share similar barcodes. The CRISPR-Cas9 system for gene editing is an ideal tool for creating a huge diversity of cellular barcodes in embryos. However, there are limitations with CRISPR-Cas9, including unpredictable indel formation and difficulties in recovering barcodes from cells. In this project, a modified CRISPR system known as prime editing will be applied in zebrafish, and utilized for lineage tracing. Prime editing allows for precise genome editing by inserting user-specified genetic sequences at a target site in the genome. I hypothesize that we can use prime editing to insert a huge library of user-specified barcodes into the genome of developing zebrafish. Because these barcodes are defined by the experimenter, they can be recovered at the end of the experiment using RNA in situ hybridization. In principle, lineage tracing with prime editing will allow us to discover the spatial arrangement of related cells in intact embryos and tissues. We hope to use lineage tracing with prime editing to understand the mechanisms of heart regeneration in zebrafish.