Lineage tracing using a Cas9-deaminase barcoding system targeting endogenous L1 elements

Abstract

Determining cell lineage and function is critical to understanding human physiology and pathology. Although advances in lineage tracing methods provide new insight into cell fate, defining cellular diversity at the mammalian level remains a challenge. Here, we develop a genome editing strategy using a cytidine deaminase fused with nickase Cas9 (nCas9) to specifically target endogenous interspersed repeat regions in mammalian cells. The resulting mutation patterns serve as a genetic barcode, which is induced by targeted mutagenesis with single-guide RNA (sgRNA), leveraging substitution events, and subsequent read out by a single primer pair. By analyzing interspersed mutation signatures, we show the accurate reconstruction of cell lineage using both bulk cell and single-cell data. We envision that our genetic barcode system will enable fine-resolution mapping of organismal development in healthy and diseased mammalian states.Lineage tracing has provided new insights into cell fate but defining cellular diversity remains a challenge. Here the authors target endogenous repeat regions in mammalian cells with cytidine deaminase fused to nCas9 to create genetic barcodes for fine-resolution mapping.