Protocol for scarless genome editing of human pluripotent stem cell based on orthogonal selective reporters

Yun Zhao; Zihang Pan; Zixuan Hong; Mengze Sun; Yi Hong; Xiaohong Peng; Xiaoxia Li; Xi Wang; Kai Wang

STAR Protocols (Jun 2024)

Protocol for scarless genome editing of human pluripotent stem cell based on orthogonal selective reporters

Yun Zhao,
Zihang Pan,
Zixuan Hong,
Mengze Sun,
Yi Hong,
Xiaohong Peng,
Xiaoxia Li,
Xi Wang,
Kai Wang

Affiliations

Yun Zhao: Department of Physiology and Pathophysiology, School of Basic Medical Sciences, State Key Laboratory of Vascular Homeostasis and Remodeling, Peking University, Beijing 100191, China; Corresponding author
Zihang Pan: Department of Physiology and Pathophysiology, School of Basic Medical Sciences, State Key Laboratory of Vascular Homeostasis and Remodeling, Peking University, Beijing 100191, China
Zixuan Hong: Department of Physiology and Pathophysiology, School of Basic Medical Sciences, State Key Laboratory of Vascular Homeostasis and Remodeling, Peking University, Beijing 100191, China
Mengze Sun: Department of Physiology and Pathophysiology, School of Basic Medical Sciences, State Key Laboratory of Vascular Homeostasis and Remodeling, Peking University, Beijing 100191, China
Yi Hong: Department of Physiology and Pathophysiology, School of Basic Medical Sciences, State Key Laboratory of Vascular Homeostasis and Remodeling, Peking University, Beijing 100191, China
Xiaohong Peng: Department of Physiology and Pathophysiology, School of Basic Medical Sciences, State Key Laboratory of Vascular Homeostasis and Remodeling, Peking University, Beijing 100191, China
Xiaoxia Li: Department of Physiology and Pathophysiology, School of Basic Medical Sciences, State Key Laboratory of Vascular Homeostasis and Remodeling, Peking University, Beijing 100191, China
Xi Wang: State Key Laboratory of Female Fertility Promotion, Clinical Stem Cell Research Center, Peking University Third Hospital, Beijing 100191, China; Corresponding author
Kai Wang: Department of Physiology and Pathophysiology, School of Basic Medical Sciences, State Key Laboratory of Vascular Homeostasis and Remodeling, Peking University, Beijing 100191, China; Corresponding author

Journal volume & issue: Vol. 5, no. 2
p. 103084

Abstract

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Summary: Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine and disease modeling. Here, we present a protocol for establishing edited hPSC cell lines utilizing visualized orthogonal selective reporters. We describe steps for constructing plasmids, carrying out cell culture and electroporation, as well as performing drug-fluorescent dual enrichment, clone screening, and cell line characterization. This protocol facilitates the achievement of single-base homozygous mutations and reporter knockins, offering a reliable approach for precision genome editing. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Published in STAR Protocols

ISSN: 2666-1667 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Science (General)
Website: https://star-protocols.cell.com/

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