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

Area of Science:

• Molecular Biology
• Developmental Biology
• Genetics

Background:

• Studying endogenous protein localization and function in vivo is difficult due to complex gene targeting methods and limited allele functionality.
• Existing techniques often require laborious genetic modifications, hindering efficient investigation of native protein behavior.

Purpose of the Study:

• To develop a versatile toolkit for probing and perturbing endogenous protein function in vivo.
• To create a system that overcomes the limitations of conventional gene targeting and monofunctional alleles.
• To enable fluorescent visualization, manipulation, and degradation of target proteins using genetically encoded affinity reagents (GEARs).

Main Methods:

• Development of a multifunctional toolkit based on genetically encoded affinity reagents (GEARs).
• Utilizing small epitopes recognized by nanobodies and single-chain variable fragments within the GEARs system.
• Implementation of a CRISPR/Cas9-based epitope tagging pipeline for generating knock-in alleles.
• Application of GEARs to study the pioneer transcription factor Nanog and planar cell polarity protein Vangl2 in zebrafish development.

Main Results:

• Demonstrated the utility of GEARs for fluorescent visualization, manipulation, and degradation of endogenous proteins in vivo.
• Successfully generated knock-in alleles using a CRISPR/Cas9-based pipeline, showcasing broad applicability.
• Examined the native behavior of Nanog and Vangl2 during early zebrafish development using the GEARs toolkit.
• Established a versatile system that circumvents challenges associated with traditional gene targeting methods.

Conclusions:

• The developed GEARs toolkit offers a flexible and adaptable system for in vivo protein studies.
• This toolkit significantly simplifies the investigation of endogenous protein localization and function.
• The GEARs system provides a valuable resource for the model organism community, facilitating research in developmental biology and beyond.