Project Summary DNA recording is a recently developed technology that allows cellular signaling events of interest to be followed over time without removing cells from their physiological context. During a DNA recording experiment, genetically engineered cells expressing the DNA recorder acquire mutations at a “recording locus” as the result of transient cellular events, without perturbing their normal phenotype. At the end of the experiment, once their eventual phenotype is known, cells can be collected and their histories determined by sequencing the recording locus. The work proposed here will lead to a DNA recorder that is not limited, as current technology is, to recording one or two cellular events per experiment, but can record dozens of events in parallel, allowing an unbiased examination of cell history in any context compatible with genetically engineered cells. In this project, the new DNA recorder will be used to study long-term effects for a cell of experiencing significant spontaneous DNA damage. Although DNA damage is very well-studied, it has not previously been possible to follow the rare cells that experience significant spontaneous damage, especially in an in vivo mammalian context. The new DNA recorder will be developed in stages: First, intracellular recording machinery, based on a pre-existing recorder, will be created for a small number of cellular events associated with the DNA damage response. Next, the fidelity of this new recording machinery will be extensively validated in cultured cells and cancer xenografts. Finally, a new recording architecture will be developed and validated, before being introduced into a transgenic mouse to identify how spontaneous DNA damage affects cell fate determination during normal development.

Project Narrative Every time cells divide, their DNA is damaged at a low level. This “endogenous” DNA damage is an important factor in normal and aberrant animal development, but because it happens at a low level over long time periods it hasn’t been possible to study in detail. I propose to develop a new tool with the resolving power to study endogenous DNA damage and other rare cellular processes over weeks to months in developing mammals.