Esophageal organoids serve as powerful systems to study epithelial lineage hierarchies and cancer biology. Gene manipulation in these organoids has traditionally involved overexpression or knockout strategies. However, CRISPR-/Cas9-based knock-in (KI) approaches now enable precise cell lineage tracing and live imaging. Here, we describe protocols to generate fluorescent KI organoids from murine esophageal epithelium by tagging Krt13 (BFP) and Sox2 (mNeon). These dual-reporter organoids allow direct monitoring of growth dynamics and differentiation trajectories. We outline CRISPR/Cas9 design, donor construction using homology-independent approaches (CRISPaint), delivery into organoid cells, enrichment and single-clone isolation, and validation by fluorescence. For organoids, homology-directed repair (HDR) can be relatively inefficient to deliver the reporter frame. Thus, we highlight the practical advantages of non-homologous end joining (NHEJ)-based methods, which enable robust, frame-accurate KI with minimal cloning. The methods outlined here can be applied broadly for cell lineage tracing, damage-response studies, and cancer modeling.

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Genetic Engineering of Esophageal Organoids: CRISPR-Based Knock-In for Cell Lineage Tracing

  • Kyung-Pil Ko,
  • Jae-Il Park

摘要

Esophageal organoids serve as powerful systems to study epithelial lineage hierarchies and cancer biology. Gene manipulation in these organoids has traditionally involved overexpression or knockout strategies. However, CRISPR-/Cas9-based knock-in (KI) approaches now enable precise cell lineage tracing and live imaging. Here, we describe protocols to generate fluorescent KI organoids from murine esophageal epithelium by tagging Krt13 (BFP) and Sox2 (mNeon). These dual-reporter organoids allow direct monitoring of growth dynamics and differentiation trajectories. We outline CRISPR/Cas9 design, donor construction using homology-independent approaches (CRISPaint), delivery into organoid cells, enrichment and single-clone isolation, and validation by fluorescence. For organoids, homology-directed repair (HDR) can be relatively inefficient to deliver the reporter frame. Thus, we highlight the practical advantages of non-homologous end joining (NHEJ)-based methods, which enable robust, frame-accurate KI with minimal cloning. The methods outlined here can be applied broadly for cell lineage tracing, damage-response studies, and cancer modeling.