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

Authors

Kyung-Pil Ko
Jae-Il Park

Language

English

Publication Date

10-11-2025

Journal

Methods in Molecular Biology

DOI

10.1007/7651_2025_669

PMID

41071231

PMCID

PMC12582399

PubMedCentral® Posted Date

11-4-2025

PubMedCentral® Full Text Version

Author MSS

Abstract

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.

Keywords

Esophageal organoid, CRISPR/Cas9, Non-homologous end joining, NHEJ, knock-in reporter system, Krt13, Sox2, BFP, mNeon

Published Open-Access

yes

Recommended Citation

Kyung-Pil Ko and Jae-Il Park, "Genetic Engineering of Esophageal Organoids: CRISPR-Based Knock-In for Cell Lineage Tracing" (2025). Faculty, Staff and Student Publications. 6383.
https://digitalcommons.library.tmc.edu/uthgsbs_docs/6383