TY - JOUR
T1 - Flow Assisted Mutation Enrichment (FAME)
T2 - A highly efficacious and efficient method to enrich Double Knockouts (DKO) after gene editing
AU - Hansen, Michael
AU - Cai, Xiaopin
AU - Bowen, Sara
AU - Largaespada, David A.
AU - Li, Ming V.
N1 - Publisher Copyright:
© 2021 Public Library of Science. All rights reserved.
PY - 2021/3
Y1 - 2021/3
N2 - 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.
AB - 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.
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U2 - 10.1371/journal.pone.0247375
DO - 10.1371/journal.pone.0247375
M3 - Article
C2 - 33661977
AN - SCOPUS:85102531032
SN - 1932-6203
VL - 16
JO - PloS one
JF - PloS one
IS - 3 March
M1 - e0247375
ER -