Raj Bushra
Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
Institute for Regenerative Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA.
Methods Mol Biol. 2025;2886:299-310. doi: 10.1007/978-1-0716-4310-5_15.
CRISPR-Cas tools have recently been adapted for cell lineage tracing during development. Combined with single-cell RNA sequencing, these methods enable scalable lineage tracing with single-cell resolution. Here, I describe, scGESTALTv2, which combines cumulative CRISPR-Cas9 editing of a lineage barcode array with transcriptional profiling via droplet-based single-cell RNA sequencing (scRNA-seq). The technique is applied in developing zebrafish brains to generate mutations in the barcode array during development. The recorded lineages along with cellular transcriptomes are then extracted via scRNA-seq to define cell relationships among thousands of profiled brain cells and dozens of cell types.
CRISPR-Cas工具最近已被应用于发育过程中的细胞谱系追踪。结合单细胞RNA测序，这些方法能够实现具有单细胞分辨率的可扩展谱系追踪。在此，我描述了scGESTALTv2，它将谱系条形码阵列的累积CRISPR-Cas9编辑与基于液滴的单细胞RNA测序（scRNA-seq）的转录谱分析相结合。该技术应用于发育中的斑马鱼大脑，以在发育过程中在条形码阵列中产生突变。然后通过scRNA-seq提取记录的谱系以及细胞转录组，以定义数千个被分析的脑细胞和数十种细胞类型之间的细胞关系。
