Abstract

Cellular barcoding using nuclease-induced DNA mutations is an effective approach that is emerging for recording biological information, including developmental lineages. We introduce the homing CRISPR system as an effective means of generating DNA barcodes with a high and scalable diversity. We further describe the implementation of this system in a mouse model with multiple genomically-integrated homing guide RNAs (hgRNAs). Each hgRNA can generate hundreds of mutant alleles which combine exponentially to create a large number of barcodes. Activating these hgRNAs at conception resulted in developmentally barcoded embryos wherein information about each cell’s lineage was recorded in its genome. We used these recordings to reconstruct early developmental lineages in the mouse and determine the order of axis specification in its brain. Our results provide an enabling and versatile platform for in vivo barcoding and lineage tracing in a mammalian model system.