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Genetically encoded affinity reagents are a toolkit for visualizing and manipulating endogenous protein function in vivo

Author

Listed:
  • Curtis W. Boswell

    (Yale University School of Medicine)

  • Caroline Hoppe

    (Yale University School of Medicine)

  • Alice Sherrard

    (Yale University School of Medicine)

  • Liyun Miao

    (Yale University School of Medicine)

  • Mina L. Kojima

    (Yale University School of Medicine)

  • Pieter Martino

    (Yale University School of Medicine
    Yale University School of Medicine)

  • Ning Zhao

    (University of Colorado-Anschutz Medical Campus)

  • Timothy J. Stasevich

    (Colorado State University
    Tokyo Institute of Technology)

  • Stefania Nicoli

    (Yale University School of Medicine
    Yale University School of Medicine)

  • Antonio J. Giraldez

    (Yale University School of Medicine
    Yale University School of Medicine
    Yale University School of Medicine)

Abstract

Probing endogenous protein localization and function in vivo remains challenging due to laborious gene targeting and monofunctional alleles. Here, we develop a multifunctional and adaptable toolkit based on genetically encoded affinity reagents (GEARs). GEARs use small epitopes recognized by nanobodies and single chain variable fragments to enable fluorescent visualization, manipulation and degradation of protein targets in vivo. Furthermore, we outline a CRISPR/Cas9-based epitope tagging pipeline to demonstrate its utility for producing knock-in alleles that have broad applications. We use GEARs to examine the native behavior of the pioneer transcription factor Nanog and the planar cell polarity protein Vangl2 during early zebrafish development. Together, this toolkit provides a versatile system for probing and perturbing endogenous protein function while circumventing challenges associated with conventional gene targeting and is broadly available to the model organism community.

Suggested Citation

  • Curtis W. Boswell & Caroline Hoppe & Alice Sherrard & Liyun Miao & Mina L. Kojima & Pieter Martino & Ning Zhao & Timothy J. Stasevich & Stefania Nicoli & Antonio J. Giraldez, 2025. "Genetically encoded affinity reagents are a toolkit for visualizing and manipulating endogenous protein function in vivo," Nature Communications, Nature, vol. 16(1), pages 1-20, December.
    • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-61003-w
    DOI: 10.1038/s41467-025-61003-w
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    References listed on IDEAS

    as
    1. Maria Jussila & Curtis W. Boswell & Nigel W. Griffiths & Patrick G. Pumputis & Brian Ciruna, 2022. "Live imaging and conditional disruption of native PCP activity using endogenously tagged zebrafish sfGFP-Vangl2," Nature Communications, Nature, vol. 13(1), pages 1-13, December.
    2. Ning Zhao & Kouta Kamijo & Philip D. Fox & Haruka Oda & Tatsuya Morisaki & Yuko Sato & Hiroshi Kimura & Timothy J. Stasevich, 2019. "A genetically encoded probe for imaging nascent and mature HA-tagged proteins in vivo," Nature Communications, Nature, vol. 10(1), pages 1-16, December.
    3. Jeremy Vicencio & Carlos Sánchez-Bolaños & Ismael Moreno-Sánchez & David Brena & Charles E. Vejnar & Dmytro Kukhtar & Miguel Ruiz-López & Mariona Cots-Ponjoan & Alejandro Rubio & Natalia Rodrigo Meler, 2022. "Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes," Nature Communications, Nature, vol. 13(1), pages 1-13, December.
    4. Brian Ciruna & Andreas Jenny & Diana Lee & Marek Mlodzik & Alexander F. Schier, 2006. "Planar cell polarity signalling couples cell division and morphogenesis during neurulation," Nature, Nature, vol. 439(7073), pages 220-224, January.
    5. Henner F. Farin & Ingrid Jordens & Mohammed H. Mosa & Onur Basak & Jeroen Korving & Daniele V. F. Tauriello & Karin de Punder & Stephane Angers & Peter J. Peters & Madelon M. Maurice & Hans Clevers, 2016. "Visualization of a short-range Wnt gradient in the intestinal stem-cell niche," Nature, Nature, vol. 530(7590), pages 340-343, February.
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