Abstract
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.
Original language | English |
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Pages (from-to) | 664-669 |
Number of pages | 6 |
Journal | Cytometry Part A |
Volume | 103 |
Issue number | 8 |
Early online date | 9 May 2023 |
DOIs | |
State | Published - Aug 2023 |
Bibliographical note
Publisher Copyright:© 2023 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.
Funding
This work was funded by the National Institutes of Health Common Fund 4D Nucleome Program grant (U01DK127422‐01).
Funders | Funder number |
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National Institutes of Health Common Fund 4D Nucleome Program | U01DK127422‐01 |
Keywords
- CRISPR Cas12a
- FACS
- cell sorting
- gene knock-in
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Dive into the research topics of 'Utilizing flow cytometry sorting signal width to enrich for cells positive to endogenous gene integration of fluorescent proteins'. Together they form a unique fingerprint.Equipment
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Cell Sorter - BD FACSAria-III with 4 lasers
Hauschner, H. (Manager)
The Mina and Everard Goodman Faculty of Life Sciences - at Bar-Ilan UniversityEquipment/facility: Equipment
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Flow Cytometry
Hauschner, H. (Manager), Shoval, I. (Manager), Grad, E. (Manager), Raiff, A. (Operator) & Knop, O. (Operator)
The Mina and Everard Goodman Faculty of Life Sciences - at Bar-Ilan UniversityEquipment/facility: Facility
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Imaging Analyzer - ImageStream®X Mk II
Yehuda, R. (Manager) & Hauschner, H. (Manager)
The Mina and Everard Goodman Faculty of Life Sciences - at Bar-Ilan UniversityEquipment/facility: Equipment