Abstract
Gene editing has become an essential tool for interrogation of gene function in biomedical research and is also a promising approach for gene therapy. Despite recent progresses, the gene-editing procedure is still a tedious process involving manually isolating large number of single cell colonies to screen for desired mutations. For diploid eukaryotic cells, there is the additional challenge to inactivate both alleles for genes-of-interest, i.e., generating double knockouts (DKOs), for the desired phenotypes or therapeutic effects. In this report, we present a novel method based on Fluorescence Assisted Cell Sorting (FACS) to enrich for DKO cells, using a cell surface marker β2-microglobulin (B2M) as a basis for negative selection. This method significantly increased percentage of DKOs in isolated cells after gene editing, and in the meantime, significantly improve the efficiency of workflow by automating colony isolation. It would greatly facilitate future biomedical research including potential gene/cell therapies.
Original language | English (US) |
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Article number | e0247375 |
Journal | PloS one |
Volume | 16 |
Issue number | 3 March |
DOIs | |
State | Published - Mar 2021 |
Bibliographical note
Publisher Copyright:© 2021 Public Library of Science. All rights reserved.
Keywords
- Flow Cytometry
- Gene Editing
- Gene Knockout Techniques
- HEK293 Cells
- Humans
PubMed: MeSH publication types
- Journal Article
- Research Support, N.I.H., Extramural
- Research Support, Non-U.S. Gov't