Utilizing flow cytometry sorting signal width to enrich for cells positive to endogenous gene integration of fluorescent proteins.
CRISPR Cas12a
FACS
cell sorting
gene knock-in
Journal
Cytometry. Part A : the journal of the International Society for Analytical Cytology
ISSN: 1552-4930
Titre abrégé: Cytometry A
Pays: United States
ID NLM: 101235694
Informations de publication
Date de publication:
08 2023
08 2023
Historique:
revised:
03
04
2023
received:
06
01
2023
accepted:
24
04
2023
medline:
22
8
2023
pubmed:
9
5
2023
entrez:
9
5
2023
Statut:
ppublish
Résumé
Endogenous gene knock-in using CRIPSR is becoming the standard for fluorescent tagging of endogenous proteins. Some protocols, particularly those that utilize insert cassettes that carry a fluorescent protein tag, can yield many types of cells with off-target insertions that have diffuse fluorescent signal throughout the whole cell in addition to scarce cells with on-target gene insertions that show the correct sub-cellular localization of the tagged protein. As such, when searching for cells with on-target integration using flow cytometry, the off-target fluorescent cells yield a high percentage of false positives. Here, we show that by changing the gating used to select for fluorescence during flow cytometry sorting, namely utilizing the width of the signal as opposed to the area, we can highly enrich for positively integrated cells. Reproducible gates were created to select even minuscule percentages of correct subcellular signal, and these parameters were validated by fluorescence microscopy. This method is a powerful tool to rapidly enhance the generation of cell lines with correctly integrated gene knock-ins encoding endogenous fluorescent proteins.
Identifiants
pubmed: 37158244
doi: 10.1002/cyto.a.24735
doi:
Substances chimiques
Proteins
0
Coloring Agents
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
664-669Subventions
Organisme : NIH HHS
ID : U01DK127422-01
Pays : United States
Commentaires et corrections
Type : UpdateOf
Informations de copyright
© 2023 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.
Références
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