CRISPR-Mediated Protein Tagging with Nanoluciferase to Investigate Native Chemokine Receptor Function and Conformational Changes.

07:00 EST 7th February 2020 | BioPortfolio

Summary of "CRISPR-Mediated Protein Tagging with Nanoluciferase to Investigate Native Chemokine Receptor Function and Conformational Changes."

G protein-coupled receptors are a major class of membrane receptors that mediate physiological and pathophysiological cellular signaling. Many aspects of receptor activation and signaling can be investigated using genetically encoded luminescent fusion proteins. However, the use of these biosensors in live cell systems requires the exogenous expression of the tagged protein of interest. To maintain the normal cellular context here we use CRISPR/Cas9-mediated homology-directed repair to insert luminescent tags into the endogenous genome. Using NanoLuc and bioluminescence resonance energy transfer we demonstrate fluorescent ligand binding at genome-edited chemokine receptors. We also demonstrate that split-NanoLuc complementation can be used to investigate conformational changes and internalization of CXCR4 and that recruitment of β-arrestin2 to CXCR4 can be monitored when both proteins are natively expressed. These results show that genetically encoded luminescent biosensors can be used to investigate numerous aspects of receptor function at native expression levels.


Journal Details

This article was published in the following journal.

Name: Cell chemical biology
ISSN: 2451-9448


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Medical and Biotech [MESH] Definitions

Protein components of the CRISPR-CAS SYSTEMS for anti-viral defense in ARCHAEA and BACTERIA. These are proteins that carry out a variety of functions during the creation and expansion of the CRISPR ARRAYS, the capture of new CRISPR SPACERS, biogenesis of SMALL INTERFERING RNA (CRISPR or crRNAs), and the targeting and silencing of invading viruses and plasmids. They include DNA HELICASES; RNA-BINDING PROTEINS; ENDONUCLEASES; and RNA and DNA POLYMERASES.

Adaptive antiviral defense mechanisms, in archaea and bacteria, based on DNA repeat arrays called CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR elements) that function in conjunction with CRISPR-ASSOCIATED PROTEINS (Cas proteins). Several types have been distinguished, including Type I, Type II, and Type III, based on signature motifs of CRISPR-ASSOCIATED PROTEINS.

The body's defense mechanism against foreign organisms or substances and deviant native cells. It includes the humoral immune response and the cell-mediated response and consists of a complex of interrelated cellular, molecular, and genetic components.

The exposure of protein to chemicals, or heat, which disrupt the non-covalent bonds and/or disulfide bonds responsible for maintaining the three-dimensional shape and activity of the native protein, while leaving the peptide bonds intact.

Immunologic method used for detecting or quantifying immunoreactive substances. The substance is identified by first immobilizing it by blotting onto a membrane and then tagging it with labeled antibodies.

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