Cells that can count how many times they have cycled would be widely applicable both as research tools to study variations in cell cycle control and as control systems for activating synthetic genetic programs after a defined delay. We propose to develop a genetic device that achieves both programmable and accurate cell cycle counting in human cells. To achieve programmability so that the maximum count can be extended at will, we will use sequence programmable CRISPR guide RNAs to record and execute each counting step. To achieve accuracy, we will arrange these steps so that two CRISPR enzymes execute alternating steps, and we will separate these steps temporally by co-opting a natural circuit by which cells limit DNA replication to once per cell cycle. At the end of this study, we will have advanced genetic counter design to a stage where counters can be employed for certain basic science applications in human cells, and will have revealed the ways in which the counter can be further developed so that it is sufficiently robust for long-term biological and therapeutic applications.
Cells that can ?count? how many times they?ve divided and execute genetic programs based on the count could serve both as basic research tools for studying the cell cycle and as devices for controlling therapeutic cells. For example, an accurate cell cycle counter would allow researchers to program engineered therapeutic cells to die after a chosen number of divisions so that they don?t transform into cancer after carrying out their task. By borrowing natural components that control the cell cycle and by exploiting sequence-programmable CRISPR guide RNA systems, we will develop a genetic device that can accurately count up to a high user-chosen number of cell cycles.
