Method for selecting cells based on CRISPR/Cas-mediated integration of a detectable tag to a target protein

    公开(公告)号:US20210403924A1

    公开(公告)日:2021-12-30

    申请号:US17362038

    申请日:2021-06-29

    Inventor: Alexander HAAS

    Abstract: Herein is reported a method for providing cells that express a fusion protein from a marker protein and a cell-endogenous target protein, consisting of the steps a) transfecting the cells i) with a Cas9-encoding plasmid additionally containing a nucleic acid which is resistant to a first selection agent, ii) with a circular donor plasmid containing a first nucleic acid which confers resistance to a second selection reagent, and a second nucleic acid which encodes the marker protein and which is flanked 3′ and 5′ by nucleic acids homologous to the integration site in the cell, iii) a suitable synthetic crRNA, and iv) a suitable synthetic tracrRNA, b) cultivating the cells in the presence of the first and the second selection reagent, and c) selecting cells which under the conditions of step b) carry out cell division, and thereby providing/producing cells that express a fusion protein of a marker protein and a cell-endogenous target protein.

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