Synthetic DNA clock: Recording of elapsed time based on Cas9-directed mutagenesis in human cells

We present synthetic biological systems that enable the recording of elapsed time over hours to weeks in DNA sequences in human cells. We lentivirally delivered self-targeting guide RNA-encoding sequences or pairs of target sequences and corresponding single-guide RNA-encoding sequences into Cas9- or chemically inducible Cas9-knockin human cells, respectively. The intact target sequence frequency decreased over time according to exponential decay models with the half-lives ranging from 48 hours to 750 days. By analyzing the DNA sequences, we measured elapsed time periods ranging from a few hours to 60 days with an average error of less than 10% in a highly reproducible manner. Application of these systems enabled recording of both the duration of a chemical exposure in cultured cells and the length of life after initiation of recording in mice in vivo. We propose that our systems could serve as “synthetic DNA clocks” that record elapsed time in DNA sequences.