SUMMARY Time is a problem for biologists, especially for those interested in cancer, stem cell, and developmental biology. Whereas cellular processes occur in real time, we most often try to understand biology by piecing together information obtained with static end-point experiments. The goal of this proposal is to expand a novel technology to provide innovative of dealing with problems posed by time in biology. The technology records a ledger of events within the cell’s own genome with CRISPR/Cas9 gene editing tools, which have been developed to execute machine-like instructions in cells. The ledger can be “read” by next generation DNA sequencing, allowing the reading many ledger entries at one time at once. The presence of multiple ledger entries on a contiguous DNA strand enables all the entries from many cells to be read at the individual cell level. The system of recording is based on combining multiple modular activities in series to develop a cascading process through sequentially activated steps; each step is encoded by a single module. We have developed and validated the essential components of individual modules, and here we propose to build and optimize series of modules linked together in pre-programmed orders. The proposed research is organized by three Specific Aims of 1) expanding the duration of stem cell lineage tracking with a 10-module long cascade, 2) record the history of Wnt signaling in individual cells in a lineage using our technology, and 3) control a ratcheting progression of cellular recording such that cellular events can be identified with a cell division number tracker. Completion of the proposal will provide a substantial new capability for biologists in interrogating the functions of biological systems.

NARRATIVE The proposed project will generate a new technology making it possible to execute multiple genetic manipulations in a pre-programmed, sequential manner in cells. This advance will provide a means of recording information about a cell’s history (i.e. where it came from, whether it was exposed to a toxin or hormone) in its genome for reading by DNA sequencing.