A temporally resolved, multiplex molecular recorder based on sequential genome editing

<jats:title>Abstract</jats:title> <jats:p> DNA is naturally well-suited to serve as a digital medium for <jats:italic>in vivo</jats:italic> molecular recording. However, DNA-based memory devices described to date are constrained in terms of the number of distinct signals that can be concurrently recorded and/or by a failure to capture the precise <jats:underline>order</jats:underline> of recorded events <jats:sup>1</jats:sup> . Here we describe DNA Ticker Tape, a general system for <jats:italic>in vivo</jats:italic> molecular recording that largely overcomes these limitations. Blank DNA Ticker Tape consists of a tandem array of partial CRISPR-Cas9 target sites, with all but the first site truncated at their 5’ ends, and therefore inactive. Signals of interest are coupled to the expression of specific prime editing guide RNAs <jats:sup>2</jats:sup> . Editing events are insertional, and record the identity of the guide RNA mediating the insertion while also shifting the position of the “write head” by one unit along the tandem array, <jats:italic>i.e.</jats:italic> sequential genome editing. In this proof-of-concept of DNA Ticker Tape, we demonstrate the recording and decoding of complex event histories or short text messages; evaluate the performance of dozens of orthogonal tapes; and construct “long tape” potentially capable of recording the order of as many as 20 serial events. Finally, we demonstrate how DNA Ticker Tape simplifies the decoding of cell lineage histories. </jats:p>