Advances in CRISPR–Cas9 genome editing have enabled the prospective tracking of cell lineages in diverse cells, tissues and lower vertebrates, such as zebrafish and axolotl, but application to mammals has been hampered by challenges arising from the complexity of mammalian development. Now, Kalhor et al. report the successful recording and reconstruction of developmental lineages in the mouse using in vivo-generated barcodes.

Credit: Redmond Durrell/Alamy

The team built on proof-of-principle concepts of the genome editing of synthetic target arrays for lineage tracing (GESTALT) technique, whereby lineage barcodes that consist of multiple Cas9 target sites accumulate unique mutations over time. The incrementally edited barcodes from thousands of cells are recovered by targeted sequencing, and lineages are reconstructed from the pattern of shared mutations among cells.

To apply this approach to mice, the authors generated the MARC1 (mouse for actively recording cells) founder mouse, which harbours 60 homing CRISPR guide RNA (hgRNA) loci in its genome, and crossed it with females constitutively expressing Cas9 protein. Upon Cas9-mediated activation, the hgRNA, which comprises a unique 10-base identifier and a spacer sequence, targets its own locus for mutation. In offspring, hgRNA loci begin accumulating lineage-specific mutations shortly after conception, with mutagenesis continuing throughout gestation. The hgRNA loci thus act as genetic barcodes as closely related cells exhibit greater similarity in mutational patterns within spacer sequences than more distant cells, enabling reconstruction of developmental lineages. Importantly, by targeting multiple sites simultaneously, the approach by Kalhor et al. ensures that mutations accumulate independently of each other. When combined, this creates the exponential diversity in barcodes that is required for the complex mammalian system.

Focusing on the first lineage segregation events in mouse, the authors were able to reconstruct accurate and robust lineage trees for the early developmental stages in four embryos. Finally, the team used developmentally barcoded mice to investigate axis development in the brain.

This study further highlights the potential of cumulative and combinatorial barcode editing to track cell lineages in whole organisms, including mammals. Beyond lineage tracing, this platform may be useful for recording cellular signals over time.