1. Biochemistry and Chemical Biology
  2. Genetics and Genomics
Download icon

A single-chain and fast-responding light-inducible Cre recombinase as a novel optogenetic switch

  1. Hélène Duplus-Bottin
  2. Martin Spichty
  3. Gérard Triqueneaux
  4. Christophe Place
  5. Philippe Emmanuel Mangeot
  6. Théophile Ohlmann
  7. Franck Vittoz
  8. Gaël Yvert  Is a corresponding author
  1. CNRS and Ecole Normale Superieure de Lyon, France
  2. INSERM and Ecole Normale Superieure de Lyon, France
Tools and Resources
  • Cited 0
  • Views 428
  • Annotations
Cite this article as: eLife 2021;10:e61268 doi: 10.7554/eLife.61268

Abstract

Optogenetics enables genome manipulations with high spatiotemporal resolution, opening exciting possibilities for fundamental and applied biological research. Here, we report the development of LiCre, a novel light-inducible Cre recombinase. LiCre is made of a single flavin-containing protein comprising the AsLOV2 photoreceptor domain of Avena sativa fused to a Cre variant carrying destabilizing mutations in its N-terminal and C-terminal domains. LiCre can be activated within minutes of illumination with blue light, without the need of additional chemicals. When compared to existing photoactivatable Cre recombinases based on two split units, LiCre displayed faster and stronger activation by light as well as a lower residual activity in the dark. LiCre was efficient both in yeast, where it allowed us to control the production of β-carotene with light, and in human cells. Given its simplicity and performances, LiCre is particularly suited for fundamental and biomedical research, as well as for controlling industrial bioprocesses.

Article and author information

Author details

  1. Hélène Duplus-Bottin

    Laboratory of Biology and Modeling of the Cell, CNRS UMR5239, CNRS and Ecole Normale Superieure de Lyon, Lyon, France
    Competing interests
    Hélène Duplus-Bottin, A patent application covering LiCre and its potential applications has been filed. Ref: FR3079832 A1 and WO2019193205. Patent applicant: CNRS; inventors: Hélène Duplus-Bottin, Martin Spichty and Gaël Yvert..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2029-5646

  2. Martin Spichty

    Laboratory of Biology and Modeling of the Cell, CNRS UMR5239, CNRS and Ecole Normale Superieure de Lyon, Lyon, France
    Competing interests
    Martin Spichty, A patent application covering LiCre and its potential applications has been filed. Ref: FR3079832 A1 and WO2019193205. Patent applicant: CNRS; inventors: Hélène Duplus-Bottin, Martin Spichty and Gaël Yvert..

  3. Gérard Triqueneaux

    Laboratory of Biology and Modeling of the Cell, CNRS UMR5239, CNRS and Ecole Normale Superieure de Lyon, Lyon, France
    Competing interests
    No competing interests declared.

  4. Christophe Place

    Laboratory of Physics, CNRS UMR5672, CNRS and Ecole Normale Superieure de Lyon, Lyon, France
    Competing interests
    No competing interests declared.

  5. Philippe Emmanuel Mangeot

    CIRI-Centre International de Recherche en Infectiologie, Inserm U1111, CNRS UMR5308, INSERM and Ecole Normale Superieure de Lyon, Lyon, France
    Competing interests
    No competing interests declared.

  6. Théophile Ohlmann

    CIRI-Centre International de Recherche en Infectiologie, Inserm U1111, CNRS UMR5308, INSERM and Ecole Normale Superieure de Lyon, Lyon, France
    Competing interests
    No competing interests declared.

  7. Franck Vittoz

    Laboratory of Physics, CNRS UMR5672, CNRS and Ecole Normale Superieure de Lyon, Lyon, France
    Competing interests
    No competing interests declared.

  8. Gaël Yvert

    Laboratory of Biology and Modeling of the Cell, CNRS UMR5239, CNRS and Ecole Normale Superieure de Lyon, Lyon, France
    For correspondence
    Gael.Yvert@ens-lyon.fr
    Competing interests
    Gaël Yvert, A patent application covering LiCre and its potential applications has been filed. Ref: FR3079832 A1 and WO2019193205. Patent applicant: CNRS; inventors: Hélène Duplus-Bottin, Martin Spichty and Gaël Yvert..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1955-4786

Funding

H2020 European Research Council (StG-281359 (SiGHT))

  • Gaël Yvert

Centre National de la Recherche Scientifique (MITI 80 Prime READGEN)

  • Gaël Yvert

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Kevin H Gardner, CUNY Advanced Science Research Center, United States

Publication history

  1. Received: July 20, 2020
  2. Accepted: February 22, 2021
  3. Accepted Manuscript published: February 23, 2021 (version 1)

Copyright

© 2021, Duplus-Bottin et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 428
    Page views
  • 96
    Downloads
  • 0
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Categories and tags

Research organisms

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology

    Complex structures of Rsu1 and PINCH1 reveal a regulatory mechanism of the ILK/PINCH/Parvin complex for F-actin dynamics

    Haibin Yang et al.
    Research Article Updated

    Communications between actin filaments and integrin-mediated focal adhesion (FA) are crucial for cell adhesion and migration. As a core platform to organize FA proteins, the tripartite ILK/PINCH/Parvin (IPP) complex interacts with actin filaments to regulate the cytoskeleton-FA crosstalk. Rsu1, a Ras suppressor, is enriched in FA through PINCH1 and plays important roles in regulating F-actin structures. Here, we solved crystal structures of the Rsu1/PINCH1 complex, in which the leucine-rich-repeats of Rsu1 form a solenoid structure to tightly associate with the C-terminal region of PINCH1. Further structural analysis uncovered that the interaction between Rsu1 and PINCH1 blocks the IPP-mediated F-actin bundling by disrupting the binding of PINCH1 to actin. Consistently, overexpressing Rsu1 in HeLa cells impairs stress fiber formation and cell spreading. Together, our findings demonstrated that Rsu1 is critical for tuning the communication between F-actin and FA by interacting with the IPP complex and negatively modulating the F-actin bundling.

    1. Biochemistry and Chemical Biology

    Controlling opioid receptor functional selectivity by targeting distinct subpockets of the orthosteric site

    Rajendra Uprety et al.
    Research Article Updated

    Controlling receptor functional selectivity profiles for opioid receptors is a promising approach for discovering safer analgesics; however, the structural determinants conferring functional selectivity are not well understood. Here, we used crystal structures of opioid receptors, including the recently solved active state kappa opioid complex with MP1104, to rationally design novel mixed mu (MOR) and kappa (KOR) opioid receptor agonists with reduced arrestin signaling. Analysis of structure-activity relationships for new MP1104 analogs points to a region between transmembrane 5 (TM5) and extracellular loop (ECL2) as key for modulation of arrestin recruitment to both MOR and KOR. The lead compounds, MP1207 and MP1208, displayed MOR/KOR Gi-partial agonism with diminished arrestin signaling, showed efficient analgesia with attenuated liabilities, including respiratory depression and conditioned place preference and aversion in mice. The findings validate a novel structure-inspired paradigm for achieving beneficial in vivo profiles for analgesia through different mechanisms that include bias, partial agonism, and dual MOR/KOR agonism.

    1. Biochemistry and Chemical Biology

    Identification of host proteins differentially associated with HIV-1 RNA splice variants

    Rachel Knoener et al.
    Tools and Resources

    HIV-1 generates unspliced (US), partially spliced (PS), and completely spliced (CS) classes of RNAs, each playing distinct roles in viral replication. Elucidating their host protein ‘interactomes’ is crucial to understanding virus-host interplay. Here, we present HyPR-MSSV for isolation of US, PS, and CS transcripts from a single population of infected CD4+ T-cells and mass spectrometric identification of their in vivo protein interactomes. Analysis revealed 212 proteins differentially associated with the unique RNA classes, including preferential association of regulators of RNA stability with US and PS transcripts and, unexpectedly, mitochondria-linked proteins with US transcripts. Remarkably, >80 of these factors screened by siRNA knockdown impacted HIV-1 gene expression. Fluorescence microscopy confirmed several to co-localize with HIV-1 US RNA and exhibit changes in abundance and/or localization over the course of infection. This study validates HyPR-MSSV for discovery of viral splice variant protein interactomes and provides an unprecedented resource of factors and pathways likely important to HIV-1 replication.