CRISPR-Cas9-mediated induction of large chromosomal inversions in human bronchial epithelial cells

Andriani Angelopoulou; Angelos Papaspyropoulos; Argyris Papantonis; Vassilis G Gorgoulis

doi:10.1016/j.xpro.2022.101257

CRISPR-Cas9-mediated induction of large chromosomal inversions in human bronchial epithelial cells

STAR Protoc. 2022 Mar 18;3(2):101257. doi: 10.1016/j.xpro.2022.101257. eCollection 2022 Jun 17.

Authors

Andriani Angelopoulou^{1

2}, Angelos Papaspyropoulos^{1

2}, Argyris Papantonis^{3

4}, Vassilis G Gorgoulis^{1

2

5

6

7}

Affiliations

¹ Molecular Carcinogenesis Group, Department of Histology and Embryology, Faculty of Medicine, National and Kapodistrian University of Athens, 11527 Athens, Greece.
² Biomedical Research Foundation, Academy of Athens, 11527 Athens, Greece.
³ Translational Epigenetics Group, Institute of Pathology, University Medical Center Göttingen, 37075 Göttingen, Germany.
⁴ Center for Molecular Medicine Cologne (CMMC), University of Cologne, 50931 Cologne, Germany.
⁵ Division of Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine & Health, University of Manchester, M20 4GJ Manchester, UK.
⁶ Center for New Biotechnologies and Precision Medicine, Medical School, National and Kapodistrian University of Athens, 11527 Athens, Greece.
⁷ Faculty of Health and Medical Sciences, University of Surrey, GU2 7YH Surrey, UK.

Abstract

The in vitro recapitulation of chromosomal rearrangements is a necessary tool for understanding malignancy at the molecular level. Here, we describe the targeted induction of a large chromosomal inversion (>3.7 Mbp) through CRISPR-Cas9-mediated genome editing. As inversions occur at low frequency following Cas9 cleavage, we provide a detailed screening approach of FACS-sorted, single-cell-derived clonal human bronchial epithelial cell (HBEC) cultures. The protocol provided is tailored to HBECs; however, it can be readily applied to additional adherent cellular models. For complete details on the use and execution of this protocol, please refer to Zampetidis et al. (2021).

Keywords: CRISPR; Cancer; Cell Biology; Cell culture; Flow Cytometry/Mass Cytometry; Molecular Biology; Sequencing; Single Cell.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Cas Systems* / genetics
Cells, Cultured
Chromosome Inversion*
Epithelial Cells
Gene Editing / methods
Humans