Massively parallel clonal analysis using CRISPR/Cas9 induced genetic scars

Abstract

3 equal contribution 4 correspondence: janphilipp.junker@mdc-berlin.de (J.P.J.), a.vanoudenaarden@hubrecht.eu (A.v.O.) A key goal of developmental biology is to understand how a single cell transforms into a full-grown organism consisting of many cells. Although impressive progress has been made in lineage tracing using imaging approaches, analysis of vertebrate lineage trees has mostly been limited to relatively small subsets of cells. Here we present scartrace, a strategy for massively parallel whole-organism lineage tracing based on Cas9 induced genetic scars in the zebrafish.The timing of each cell division and the fate of the progeny define the lineage of an organism. Analysis of the lineage history of cell populations can reveal the developmental origin and the clonality of cell populations 1 . Genetically encoded fluorescent proteins are widely used as lineage markers 2,3 , but due to limited spectral resolution this approach has mostly been restricted to tracking the lineage of a relatively small subset of cells. Recent progress in live imaging has allowed for following many individual cells over time in optically transparent samples such as early fly and zebrafish embryos 4,5 . Nevertheless, direct observation of all cell divisions is generally only possible at the earliest developmental stages. RNA sequencing has emerged as a powerful method for systematic expression profiling of single cells and for computational inference of differentiation dynamics 6-8 . However, our ability to harness the enormous multiplexing capacity of high-throughput sequencing for lineage tracing has so far been lagging behind, despite pioneering studies in the hematopoietic system based on viral barcoding 9,10 or transposon

Single-cell mapping of gene expression landscapes and lineage in the zebrafish embryo

“…4A). TracerSeq offers an advantage over related Cas9-based approaches (19, 20), which can generate identical edits and/or large barcode deletions in independent lineages at non-trivial frequencies. By contrast, TracerSeq barcodes are uniformly distributed over a large sequence space (e.g., 4 20 = 10 12 unique sequences), facilitating straightforward calling of genetic clones (fig.…”

Clonal analysis of lineage fate in native haematopoiesis

“…Finally, our results are still consonant with the idea that adult LT-HSCs have a limited lympho-myelo-erythroid output during steady-state 11,25 , though this finding has been debated 26 . Future work with second generation cell barcoding strategies 27,28 in combination with Cre-based labelling will be needed to elucidate full lineage histories and determine the mechanisms of fate restriction.…”

Endogenous CRISPR arrays for scalable whole organism lineage tracing

“…By identifying endogenous CRISPR arrays with appropriate properties we demonstrate that it is not necessary to generate a transgenic animal to use CRISPR arrays for lineage tracing as in previous studies [15][16][17][18][19] . And importantly our technique also does not depend on single-cell sequencing.…”

“…Zygotic injection of the CRISPR machinery that targets that array therefore generates the diversity at that location which can be readily deep sequenced and used for lineage tracing. A second approach generates lineaging barcodes by targeting the same sequence in single or multiple repeats of a transgenic fluorescent protein genes [16][17][18][19] . However, both of these approaches suffer from the drawback of requiring the generation of a transgenic animal.…”