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.
Highlights
Determining cell lineage and function is critical to understanding human physiology and pathology
We hypothesized that clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-induced double-strand breaks (DSBs) in these numerous endogenous interspersed sites present in the genome would result in the loss of useful information
Because the tree reconstruction efficiency was better with single-guide RNA (sgRNA)-3 than sgRNA-1, we focused on sgRNA-3 only and chose HeLa cells for the single-cell level lineage tracing for the ease of isolating single-cells
Summary
Determining cell lineage and function is critical to understanding human physiology and pathology. Researchers have shown that the editing of multiple endogenous retrovirus genes can be achieved without altering normal development[16] Inspired by this evidence, we reasoned that multiple target regions in various interspersed sites in repeat elements could serve as barcodes for lineage analysis. We used the recently proposed base editing method involving nickase Cas[9] (nCas9) fused with cytidine deaminase (referred to as BE3 in previous literature[17] but hereafter referred to as targeted deaminase), which converts C:G base pairs to T:A base pairs in the 4–8 nucleotide region from the protospacer adjacent motif (PAM) on the distal side of the protospacer sequence without inducing DSBs. we developed a new cellular barcoding method for lineage tracing using nCas[9] fused with cytidine deaminase to target the long interspersed nuclear element-1 (L1) in the genome. We expect that our genetic barcoding system could provide insight into normal cell development as well as molecular pathology
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