Journal Article

Large-scale reconstruction of cell lineages using single-cell readout of transcriptomes and CRISPR–Cas9 barcodes by scGESTALT

Gagnon, James A, Raj, Bushra, Schier, Alexander F

2018

نظرة عامة

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.

• الناشر: Nature Publishing Group
• موضوع: Bar codes / Brain / Branches / CRISPR / Danio rerio / Editing / Embryogenesis / Embryonic growth stage / Gene sequencing / Genome editing / Genomes / Regeneration / Ribonucleic acid / RNA / Transcription / Zebrafish