(225b) Sensitive Image-Based Readout of DNA Barcodes Enables Cell Lineage and History Recording

Multicellular development depends on the molecular history of individual cells and their descendants, but these histories are usually inaccessible to direct observation. Synthetic DNA barcodes allow us to reveal clonal relationship, lineage history, and even molecular trajectory of individual cells over time. Therefore, they can be the key to a wide variety of otherwise intractable questions. However, readout of barcodes presents a tradeoff: Sequencing methods can analyze short barcodes containing a high density of sequence variants, but necessarily lose spatial information. Conversely, imaging methods preserve spatial structure, but can only distinguish lower density sequence features. Here, I present a system for sensitive readout of DNA barcodes that makes high density barcoding and synthetic recording compatible with imaging readout. This system enables reliable in situ detection of barcodes as short as 20bp. By concatenating these barcodes, we made combinatorial lentiviral libraries to investigate clonal relationship of cells in developing nervous system. Further, we showed that probe competition enables precise discrimination of single nucleotide variations in the barcodes. This allowed us to make synthetic memory arrays that record information as precise mutations made by CRISPR base editors and can be readout by microscopy in individual cells. We are now applying these technologies to improve our understanding of cell fate specification in mammalian retina. Decades of study have revealed many aspects of retinal development such as broad developmental potential of retinal progenitors and heterogeneity in their clonal composition. Nevertheless, it remains unclear how the regular structure of retina is built by clones with mostly random composition. Our goal is to make a generalizable system that can comprehensively map the clonal composition and lineage structure in the retina. Combined with multiplexed in situ transcriptional profiling, this system will allow us to link information about lineage, cell fate, and spatial organization of individual retinal cells.