Isolation of gene-edited cells via knock-in of short glycophosphatidylinositol-anchored epitope tags

Anastasia Zotova, Alexey Pichugin, Anastasia Atemasova, Ekaterina Knyazhanskaya, Elena Lopatukhina, Nikita Mitkin, Ekhson Holmuhamedov, Marina Gottikh, Dmitry Kuprash, Alexander Filatov, Dmitriy Mazurov

Research output: Contribution to journalArticlepeer-review

13 Scopus citations

Abstract

We describe Surface Oligopeptide knock-in for Rapid Target Selection (SORTS), a novel method to select mammalian cells with precise genome modifications that does not rely on cell cloning. SORTS is designed to disrupt the target gene with an expression cassette encoding an epitope tag embedded into human glycophosphatidylinositol (GPI)-anchored protein CD52. The cassette is very short, usually less than 250 nucleotides, which simplifies donor DNA construction and facilitates transgene integration into the target locus. The chimeric protein is then expressed from the target promoter, processed and exposed on the plasma membrane where it serves as a marker for FACS sorting with tag-specific antibodies. Simultaneous use of two different epitope tags enables rapid isolation of cells with biallelic knock-ins. SORTS can be easily and reliably applied to a number of genome-editing problems such as knocking out genes encoding intracellular or secreted proteins, protein tagging and inactivation of HIV-1 provirus.

Original languageEnglish (US)
Article number3132
JournalScientific reports
Volume9
Issue number1
DOIs
StatePublished - Dec 1 2019
Externally publishedYes

Bibliographical note

Funding Information:
We thank Mikhail Pashenkov from the Institute of Immunology (Moscow) for helpful discussion and critical reading of the manuscript. Most of the experiments on design and evaluation of GPI-tags (Figures 1–2), KO selection (Figure 3a–d) and sm-AID tagging (Figure 4a,b) were supported by grant 18-14-00333 from Russian Science Foundation. In frame epitope expression (Figure 3 e-g) and HIV-1 studies (Figure 5) were funded by grant 18-29-07052 from Russian Foundation for Basic Research. Experiments on Ku70-smAID inducible degradation (Figure 4c,d) were supported by grant 17-14-01107 from Russian Science Foundation. Some FACS experiments were funded by the Program of fundamental research for state academies for 2013-2020, research topic 01201363823, by grants 18-04-01016 and 18-34-00712 from Russian Foundation for Basic Research, and by project 14.Z50.31.0028 from Russian Ministry of Education and Science.

Publisher Copyright:
© 2019, The Author(s).

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