Genetically encoded affinity reagents are a toolkit for visualizing and manipulating endogenous protein function in vivo
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 b...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-07-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-61003-w |
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| _version_ | 1849402149046321152 |
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| author | Curtis W. Boswell Caroline Hoppe Alice Sherrard Liyun Miao Mina L. Kojima Pieter Martino Ning Zhao Timothy J. Stasevich Stefania Nicoli Antonio J. Giraldez |
| author_facet | Curtis W. Boswell Caroline Hoppe Alice Sherrard Liyun Miao Mina L. Kojima Pieter Martino Ning Zhao Timothy J. Stasevich Stefania Nicoli Antonio J. Giraldez |
| author_sort | Curtis W. Boswell |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-66bc6b1c6d0e40caae87ed6172ab24c2 |
| institution | Kabale University |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-66bc6b1c6d0e40caae87ed6172ab24c22025-08-20T03:37:37ZengNature PortfolioNature Communications2041-17232025-07-0116112010.1038/s41467-025-61003-wGenetically encoded affinity reagents are a toolkit for visualizing and manipulating endogenous protein function in vivoCurtis W. Boswell0Caroline Hoppe1Alice Sherrard2Liyun Miao3Mina L. Kojima4Pieter Martino5Ning Zhao6Timothy J. Stasevich7Stefania Nicoli8Antonio J. Giraldez9Department of Genetics, Yale University School of MedicineDepartment of Genetics, Yale University School of MedicineDepartment of Genetics, Yale University School of MedicineDepartment of Genetics, Yale University School of MedicineDepartment of Genetics, Yale University School of MedicineDepartment of Genetics, Yale University School of MedicineDepartment of Biochemistry and Molecular Genetics, University of Colorado-Anschutz Medical CampusDepartment of Biochemistry and Molecular Biology, Colorado State UniversityDepartment of Genetics, Yale University School of MedicineDepartment of Genetics, Yale University School of MedicineAbstract 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.https://doi.org/10.1038/s41467-025-61003-w |
| spellingShingle | Curtis W. Boswell Caroline Hoppe Alice Sherrard Liyun Miao Mina L. Kojima Pieter Martino Ning Zhao Timothy J. Stasevich Stefania Nicoli Antonio J. Giraldez Genetically encoded affinity reagents are a toolkit for visualizing and manipulating endogenous protein function in vivo Nature Communications |
| title | Genetically encoded affinity reagents are a toolkit for visualizing and manipulating endogenous protein function in vivo |
| title_full | Genetically encoded affinity reagents are a toolkit for visualizing and manipulating endogenous protein function in vivo |
| title_fullStr | Genetically encoded affinity reagents are a toolkit for visualizing and manipulating endogenous protein function in vivo |
| title_full_unstemmed | Genetically encoded affinity reagents are a toolkit for visualizing and manipulating endogenous protein function in vivo |
| title_short | Genetically encoded affinity reagents are a toolkit for visualizing and manipulating endogenous protein function in vivo |
| title_sort | genetically encoded affinity reagents are a toolkit for visualizing and manipulating endogenous protein function in vivo |
| url | https://doi.org/10.1038/s41467-025-61003-w |
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