Endogenous CRISPR arrays for scalable whole organism lineage tracing

Abstract

The last 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 which completely avoids the need for prior genetic engineering, by identifying endogenous CRISPR 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 arrays, demonstrating their utility for lineage tracing. Our technique thus can produce deep and broad lineages in vivo, while removing the dependence on genetic engineering, and also avoiding the need for single-cell analysis.

High-throughput dense reconstruction of cell lineages

“…This strategy avoids the current limitations of genetic engineering, which include low cargo capacity and difficulty in obtaining high copy number integrations. Endogenous target arrays suitable for lineage tracing have been identified in the zebrafish and mouse genomes by James Cotterell from the group of James Sharpe [18]. As in GESTALT, they rely on the use of Cas9 nuclease to edit compact arrays of targets, but interestingly in this case they found fewer inter-target deletions, possibly due to lower target sequence similarities.…”