CRISPR-Cas9 介导的人支气管上皮细胞中大型染色体倒位的诱导。

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

机构信息

Molecular Carcinogenesis Group, Department of Histology and Embryology, Faculty of Medicine, National and Kapodistrian University of Athens, 11527 Athens, Greece.

Biomedical Research Foundation, Academy of Athens, 11527 Athens, Greece.

出版信息

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

DOI:10.1016/j.xpro.2022.101257

PMID:35330963

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8938320/

Abstract

The 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).

摘要

染色体重排的综述是从分子水平理解恶性肿瘤的必要工具。在这里，我们描述了通过 CRISPR-Cas9 介导的基因组编辑靶向诱导大的染色体倒位（>3.7 Mbp）。由于 Cas9 切割后倒位的发生频率较低，我们提供了一种详细的筛选方法，用于 FACS 分选的单细胞衍生的人支气管上皮细胞（HBEC）培养物的克隆。提供的方案针对 HBEC 进行了定制；但是，它可以很容易地应用于其他贴壁细胞模型。有关此方案使用和执行的完整详细信息，请参阅 Zampetidis 等人。（2021 年）。