Generation of ASCL1-mCherry knock-in reporter in human embryonic stem cell line, WAe001-A-2E, using CRISPR/Cas9-based gene targeting

Shisheng Jiang; Tiankai Dai; Qian Li; Tao Xu; Wei Zhang; Jiaqi Sun; Huisheng Liu

doi:10.1016/j.scr.2024.103500

Generation of ASCL1-mCherry knock-in reporter in human embryonic stem cell line, WAe001-A-2E, using CRISPR/Cas9-based gene targeting

Stem Cell Res. 2024 Oct:80:103500. doi: 10.1016/j.scr.2024.103500. Epub 2024 Jul 24.

Authors

Shisheng Jiang¹, Tiankai Dai², Qian Li³, Tao Xu¹, Wei Zhang¹, Jiaqi Sun¹, Huisheng Liu⁴

Affiliations

¹ School of Biomedical Engineering, Guangzhou Medical University, Guangzhou 510180, China; Guangzhou National Laboratory, Guangzhou 510005, China.
² College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China; Guangzhou National Laboratory, Guangzhou 510005, China.
³ Guangzhou National Laboratory, Guangzhou 510005, China.
⁴ School of Biomedical Engineering, Guangzhou Medical University, Guangzhou 510180, China; College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China; Guangzhou National Laboratory, Guangzhou 510005, China; Bioland Laboratory, Guangzhou 510005, China. Electronic address: [email protected].

PMID: 39059080
DOI: 10.1016/j.scr.2024.103500

Abstract

Achaete-Scute Complex Homolog 1 (ASCL1) is a key regulator in the development and function of the nervous system, particularly in the process of neuronal and neuroendocrine cell differentiation. By employing the CRISPR/Cas9 system, we successfully established an ASCL1-mCherry knock-in human embryonic stem cell (hESC) line by inserting a P2A-mCherry fragment at the ASCL1 locus. The mCherry reporter effectively demonstrated the expression level of endogenous ASCL1 during the process of inducing pulmonary neuroendocrine cells (PNECs) from hESC. This reporter cell line holds significant value as a research tool for investigating the process of lung neuroendocrine cell differentiation, conducting drug screening, and exploring the underlying mechanisms of lung diseases associated with PNECs dysfunction.

MeSH terms

Basic Helix-Loop-Helix Transcription Factors* / genetics
Basic Helix-Loop-Helix Transcription Factors* / metabolism
CRISPR-Cas Systems*
Cell Differentiation
Cell Line
Gene Knock-In Techniques
Gene Targeting*
Genes, Reporter
Human Embryonic Stem Cells* / cytology
Human Embryonic Stem Cells* / metabolism
Humans
Red Fluorescent Protein

Substances

Basic Helix-Loop-Helix Transcription Factors
ASCL1 protein, human
Red Fluorescent Protein