Nature protocolsOct 25, 2018

Tracking cell family trees using single-cell gene activity and CRISPR barcodes

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Mar 12, 2026

Abstract

Generating transgenic zebrafish takes 6 months.

The method scGESTALT combines CRISPR-Cas9 editing with single-cell RNA sequencing to trace lineage relationships among diverse cell types.
Lineage information is captured across multiple timepoints, facilitating the reconstruction of complex lineage trees.
The technique allows for the analysis of thousands of cellular transcriptomes concurrently with lineage tracing.
The process of barcode editing and creating single-cell libraries requires 7 days of hands-on effort.
This approach may be applicable in various biological systems, including development, regeneration, or disease contexts.

Simplified

Lineage relationships among the large number of heterogeneous cell types generated during development are difficult to reconstruct in a high-throughput manner. We recently established a method, scGESTALT, that combines cumulative editing of a lineage barcode array by CRISPR-Cas9 with large-scale transcriptional profiling using droplet-based single-cell RNA sequencing (scRNA-seq). The technique generates edits in the barcode array over multiple timepoints using Cas9 and pools of single-guide RNAs (sgRNAs) introduced during early and late zebrafish embryonic development, which distinguishes it from similar Cas9 lineage-tracing methods. The recorded lineages are captured, along with thousands of cellular transcriptomes, to build lineage trees with hundreds of branches representing relationships among profiled cell types. Here, we provide details for (i) generating transgenic zebrafish; (ii) performing multi-timepoint barcode editing; (iii) building scRNA-seq libraries from brain tissue; and (iv) concurrently amplifying lineage barcodes from captured single cells. Generating transgenic lines takes 6 months, and performing barcode editing and generating single-cell libraries involve 7 d of hands-on time. scGESTALT provides a scalable platform to map lineage relationships between cell types in any system that permits genome editing during development, regeneration, or disease.

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Competing interests

COMPETING INTERESTS. The authors declare no competing interests.

PubMed

Tracking cell family trees using single-cell gene activity and CRISPR barcodes

Abstract

Full Text

Funding

Competing interests

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