A method for CRISPR/Cas9-induced genetic barcoding and lineage tracing in sheep

Sheep development involves continuous dynamic processes in which cells propagate, differentiate and orchestrate. However, the method for tracing cell fate during sheep (Ovis aries) embryogenesis, cell differentiation and tissue regeneration remains largely undeveloped. Here, we developed a CRISPR/Cas9-based lineage barcode recording method that directly acts on target cells. With this method, several contiguous CRISPR/Cas9 targeting arrays were synthesized and introduced into multiple loci in the sheep genome using the PiggyBac transposon vector to form the barcode region. Cas9 integrated at the Rosa26 gene locus was used to generate edits in the barcode region at multiple timepoints during early sheep embryonic development. We detected multiple integrated barcodes (intBCs) that were stably inherited in the developing embryos, confirming that the method can generate heritable clonal markers. The method could enable lineage tracing in sheep when combined with single-cell sequencing technologies. Our method establishes a foundation for ruminant lineage tracing technology by combining PiggyBac transposons and CRISPR/Cas9 gene editing tools, providing a new platform for analyzing sheep embryonic development, organ regeneration, and disease mechanisms.