Long-Term DNA Recording with Ordered Insertion Mutations

Mammalian development involves a pattern of disparate but coordinated fate changes undergone by millions to billions of cells over days to years. DNA recording is a technique that has the potential to illuminate these processes at the appropriate scale and depth, by making durable changes in the genome that reflect transient events in the cell’s history. Recently, we published the development of a DNA recorder that writes new DNA, by inserting random nucleotides at a single locus in multiple rounds, such that the order of the nucleotides at the locus reflects the order in which they were added. Our system, called CHYRON (Cell HistorY Recording by Ordered iNsertion), uses Cas9 nuclease, a self-targeting guide (stg)RNA, and the template-independent DNA polymerase TdT. CHYRON records an average of 14.6 bits per recording site over an average of three rounds, but its recording duration is limited because ~25% of edits lead to information-corrupting deletion mutations, which compound over time, and the desired insertion mutations increase the length of the stgRNA, which reduces its activity. Now, we have created a new DNA recording architecture, peCHYRON, that maintains the strengths of CHYRON – ordered, information-dense recording – but (1) drastically reduces the rate of information loss through off-pathway mutations, (2) is in principle compatible with tens of rounds of recording, and (3) can in principle record at least tens of orthogonal inputs. In each round of peCHYRON recording, prime editor makes one single-strand nick to introduce an insertion mutation that includes both a “signature” sequence that constitutes the record and a “propagation sequence” that enables the next step of recording. In a peCHYRON stable cell line, at least 97% of edits in each round were the desired, information-preserving insertion mutations, and editing efficiency remained constant over at least the first five rounds of recording.