Endogenous CRISPR/Cas9 arrays for scalable whole-organism lineage tracing

Cotterell, James; Vila-Cejudo, Marta; Batlle Morera, Laura; Sharpe, James

doi:http://dx.doi.org/10.1242/dev.184481

Endogenous CRISPR/Cas9 arrays for scalable whole-organism lineage tracing

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Citació

Cotterell J, Vila-Cejudo M, Batlle-Morera L, Sharpe J. Endogenous CRISPR/Cas9 arrays for scalable whole-organism lineage tracing. Development. 2020 May 12; 147(9): dev184481. DOI: 10.1242/dev.184481

Resum

The past decade has seen a renewed appreciation of the central importance of cellular lineages to many questions in biology (especially organogenesis, stem cells and tumor biology). This has been driven in part by a renaissance in genetic clonal-labeling techniques. Recent approaches are based on accelerated mutation of DNA sequences, which can then be sequenced from individual cells to re-create a 'phylogenetic' tree of cell lineage. However, current approaches depend on making transgenic alterations to the genome in question, which limit their application. Here, we introduce a new method that completely avoids the need for prior genetic engineering, by identifying endogenous CRISPR/Cas9 target arrays suitable for lineage analysis. In both mouse and zebrafish, we identify the highest quality compact arrays as judged by equal base composition, 5' G sequence, minimal likelihood of residing in the functional genome, minimal off targets and ease of amplification. We validate multiple high-quality endogenous CRISPR/Cas9 arrays, demonstrating their utility for lineage tracing. Our pragmatically scalable technique thus can produce deep and broad lineages in vivo, while removing the dependence on genetic engineering.