Recording molecular information to genomic DNA is a powerful means of investigating topics ranging from multicellular development to cancer evolution. With molecular recording based on genome editing, events such as cell divisions and signaling pathway activity drive specific alterations in a cell's DNA, marking the genome with information about a cell's history that can be read out after the fact. Although genome editing has been used for molecular recording, capturing the temporal relationships among recorded events in mammalian cells remains challenging. The DNA Typewriter system overcomes this limitation by leveraging prime editing to facilitate sequential insertions to an engineered genomic region. DNA Typewriter includes three distinct components: DNA Tape as the 'substrate' to which edits accrue in an ordered manner, the prime editor enzyme, and prime editing guide RNAs, which program insertional edits to DNA Tape. In this protocol, we describe general design considerations for DNA Typewriter, step-by-step instructions on how to perform recording experiments by using DNA Typewriter in HEK293T cells, and example scripts for analyzing DNA Typewriter data ( https://doi.org/10.6084/m9.figshare.22728758 ). This protocol covers two main applications of DNA Typewriter: recording sequential transfection events with programmed barcode insertions by using prime editing and recording lineage information during the expansion of a single cell to many. Compared with other methods that are compatible with mammalian cells, DNA Typewriter enables the recording of temporal information with higher recording capacities and can be completed within 4-6 weeks with basic expertise in molecular cloning, mammalian cell culturing and DNA sequencing data analysis.
