Utilizing flow cytometry sorting signal width to enrich for cells positive to endogenous gene integration of fluorescent proteins

Gabriel P. Faber, Hagit Hauschner, Mohammad K. Atrash, Liat Bilinsky, Yaron Shav-Tal

Research output: Contribution to journalArticlepeer-review

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 languageEnglish
Pages (from-to)664-669
Number of pages6
JournalCytometry. Part A : the journal of the International Society for Analytical Cytology
Volume103
Issue number8
Early online date9 May 2023
DOIs
StatePublished - 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).

FundersFunder number
National Institutes of Health Common Fund 4D Nucleome ProgramU01DK127422‐01

    Keywords

    • CRISPR Cas12a
    • FACS
    • cell sorting
    • gene knock-in

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