Simultaneous lineage tracing and cell-type identification using CRISPR–Cas9-induced genetic scars


A key goal of developmental biology is to understand how a single cell is transformed into a full-grown organism comprising many different cell types. Single-cell RNA-sequencing (scRNA-seq) is commonly used to identify cell types in a tissue or organ1. However, organizing the resulting taxonomy of cell types into lineage trees to understand the developmental origin of cells remains challenging. Here we present LINNAEUS (lineage tracing by nuclease-activated editing of ubiquitous sequences)—a strategy for simultaneous lineage tracing and transcriptome profiling in thousands of single cells. By combining scRNA-seq with computational analysis of lineage barcodes, generated by genome editing of transgenic reporter genes, we reconstruct developmental lineage trees in zebrafish larvae, and in heart, liver, pancreas, and telencephalon of adult fish. LINNAEUS provides a systematic approach for tracing the origin of novel cell types, or known cell types under different conditions.

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Figure 1: Using the CRISPR–Cas9 system for massively parallel single-cell lineage tracing.
Figure 2: Computational reconstruction of lineage trees on the single-cell level.
Figure 3: Single-cell lineage analysis of adult organs reveals hierarchies of cell fate decisions.


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We thank R. Opitz, M. Guedes Simoes, D. Panakova, T. Durovic, and J. Ninkovic for help with cell-type identification. We also acknowledge support by MDC/BIMSB core facilities (zebrafish, genomics, bioinformatics), and we thank J. Richter for help with zebrafish experiments. Work in J.P.J.'s laboratory was funded by a European Research Council Starting Grant (ERC-StG 715361 SPACEVAR), a Fondation Leducq Transatlantic Networks Grant (16CVD03), and a Helmholtz Incubator grant (Sparse2Big ZT-I-0007). B.H. was supported by a PhD fellowship from Studienstiftung des deutschen Volkes.

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J.P.J., B.S. and B.H. conceived and designed the project. B.H., N.M. and S.J. optimized dissection and dissociation protocols. B.H. developed the experimental approach for combined scar and transcriptome detection, and B.H. and N.M. performed experiments. B.S. developed computational methods and analyzed the data, with support by P.O.-C. J.P.J. and N.N. guided experiments, and J.P.J. guided analysis. J.P.J. and B.S. wrote the manuscript, with input from all other authors. All authors discussed and interpreted results.

Corresponding author

Correspondence to Jan Philipp Junker.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–20, Supplementary Table 1 and Supplementary Note 1 (PDF 13066 kb)

Life Sciences Reporting Summary (PDF 170 kb)

Supplementary Dataset 1

Technical information for all sequenced libraries (XLSX 10 kb)

Supplementary Dataset 2

Cell type information for 5 dpf larvae (XLSX 40 kb)

Supplementary Dataset 3

Scar detection statistics for 5 dpf larvae (XLSX 14 kb)

Supplementary Dataset 4

Cell type information for adult organs (XLSX 40 kb)

Supplementary Dataset 5

Scar detection statistics for adult organs (XLSX 13 kb)

Supplementary Dataset 6

Statistics connection enrichment analysis (XLSX 146 kb)

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Spanjaard, B., Hu, B., Mitic, N. et al. Simultaneous lineage tracing and cell-type identification using CRISPR–Cas9-induced genetic scars. Nat Biotechnol 36, 469–473 (2018).

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