Mammalian cells that record their own history

On short timescales, the flow of information from DNA to cellular function is one-way. Technologies that reverse this flow by transferring non-genetic information into mutations in DNA would considerably advance how we study biology: they would allow cells to capture transient experiences, such as lineage relationships or environmental signals, into a durable and sequenceable DNA record in vivo. We have developed a synthetic locus in the genome of human cells that controllably accumulates insertion mutations in the presence of Streptococcus pyogenes Cas9 and terminal deoxynucleotidyl transferase (TdT). The locus expresses a self-targeting guide RNA that directs Cas9 to cut the locus itself, yielding a double stand break at which TdT inserts random nucleotides. Cycles of cutting, nucleotide insertion, and repair cause continuous and directional insertional mutagenesis at the locus, reminiscent of a ticker-tape. We describe the design and characterization of our “ticker-tape” locus (TTL) and demonstrate its use in recording two broad categories of non-genetic biological information, lineage and environment. When operating constitutively, the sequence of the TTL documents descent. When operating conditionally, the length of the TTL reflects exposure to the condition at single-cell resolution; we have successfully used the TTL as an analog recorder of the hypoxic response. In its current state, the TTL should have broad applications in cell, developmental, and cancer biology. Moreover, its distinguishing feature of ordered insertional mutagenesis should enable full single-cell-resolution lineage tracing in complex tumors and animals with further developments that we outline.