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

The past decade has seen a renewed appreciation of the centralimportance of cellular lineages to many questions in biology(especially organogenesis, stem cells and tumor biology). This hasbeen driven in part by a renaissance in genetic clonal-labelingtechniques. Recent approaches are based on accelerated mutationof DNA sequences, which can then be sequenced from individualcells to re-create a 'phylogenetic' tree of cell lineage. However,current approaches depend on making transgenic alterations to thegenome in question, which limit their application. Here, we introduce anew method that completely avoids the need for prior geneticengineering, by identifying endogenous CRISPR/Cas9 target arrayssuitable for lineage analysis. In both mouse and zebrafish, we identifythe highest quality compact arrays as judged by: equal basecomposition, 5' G sequence, minimal likelihood of residing in thefunctional genome, minimal off targets and ease of amplification. Wevalidate multiple high-quality endogenous CRISPR/Cas9 arrays,demonstrating their utility for lineage tracing. Our pragmaticallyscalable technique thus can produce deep and broad lineagesin vivo, while removing the dependence on genetic engineering.