PROJECT SUMMARY Just as gene and protein expression are common characteristics to identify a cell, lineage is an important aspect of cell identity. In the past, lineage tracing has been used to determine what cells arise from a specific cell type, as defined by the expression of a cell-type-specific gene; however, the establishment of single-cell genomics techniques has ushered in lineage tracing at single-cell resolution. New technologies for lineage tracing can track the progeny of single cells, regardless of their initial gene expression. The Morris lab has developed a single-cell lineage tracing (scLT) method, called CellTagging. CellTagging demonstrated the ability of scLT approaches to identify similarities in cells based on lineage and offer mechanistic insights to cell reprogramming. CellTagging and other virus-based scLT technologies still present limitations, though, in that they require multiple transductions to increase lineage resolution, and may fail to capture biologically relevant bifurcation events due to cell labeling at discrete time points. These technologies are also subject to transgene silencing in certain cell models, such as iPSC-derived organoids, rendering them ineffective for use in many models of development and disease. To overcome these limitations, it is necessary to develop new scLT tools that can be applied without repeated manipulation of cells and be used in iPSC differentiation and reprogramming systems without silencing hindering the readout of lineage information. Here, I propose to utilize a CRISPR-Cas12a-guided cytidine deaminase as a method to continuously record heritable lineage data through targeted cytidine to thymine editing. I have developed and validated the ability of a novel CRISPR-Cas12a-guided cytidine deaminase to accrue base edits on a targeted synthetic DNA region over time in vitro and recovered these synthetic sequences via single-cell RNA-sequencing (scRNA-seq). These two outcomes are a promising proof- of-concept that scLT can be performed with these accrued single base edits. Here, I propose to (1) increase the resolution of CellTagging to capture bifurcation events, using this novel DNA editor to constantly edit single bases in a targeted editing region (TER) and dispense with the need for multiple transductions, and (2) integrate this base editor system into a safe harbor locus within an iPSC line to shield the transgenic components of the technology from silencing, validating this approach in kidney organoid differentiation. My proposed developments of the CellTagging technology increase the potential for discovery because they can be broadly applied to model systems that are either not amenable to multiple manipulations or are prone to transgene silencing. By making all plasmids, cell lines, protocols, and analysis tools for these systems publicly available, I aim to provide a valuable resource across several areas of cell biology. These resources will provide an experimental toolki...