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Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

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Expanding the Chemogenetic Toolbox by Circular Permutation.

Yi-Tsang Lee¹, Lian He¹, Yubin Zhou²

¹Center for Translational Cancer Research, Institute of Biosciences and Technology, Texas A&M University, Houston, TX 77030, USA.

Journal of Molecular Biology

|April 12, 2020

Summary

This summary is machine-generated.

Researchers developed cpRAPID, a novel chemogenetic tool using circular permutation for controllable protein interactions. This system enhances molecular engineering and synthetic biology applications by enabling precise control over cellular processes.

Keywords:

CRISPR antibody engineering chemogenetics nanobody synthetic biology

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Area of Science:

Synthetic Biology
Molecular and Cellular Engineering
Biotechnology

Background:

Chemogenetics offers precise control over cellular functions using small molecules.
Existing chemogenetic systems have limitations in activation kinetics and dynamics.
Expanding the toolbox for molecular and cellular engineering is crucial for synthetic biology.

Purpose of the Study:

To design and characterize cpRAPID, a novel circularly permuted rapamycin-inducible dimerization system.
To demonstrate the utility of cpRAPID for controlling protein localization and gene expression.
To showcase cpRAPID's application in developing a chemically switchable split nanobody system.

Main Methods:

Circular permutation of the FKBP12-rapamycin binding domain (FRB) to create cpFRB.
Development of cpFRB-FKBP pairs with tunable activation kinetics.
Integration of cpRAPID with CRISPR activation (CRISPRa) for inducible gene expression.
Construction of a chemically switchable split nanobody (Chessbody) using cpRAPID.

Main Results:

cpRAPID enables chemical-controllable subcellular redistribution of proteins.
The system allows for inducible transcriptional activation when coupled with CRISPRa.
cpRAPID facilitated the creation of Chessbody for ligand-gated antigen recognition.
Circular permutation provides diverse activation kinetics and dynamics.

Conclusions:

Circular permutation is a powerful strategy for diversifying chemogenetic tools.
cpRAPID expands the capabilities of synthetic biology for molecular and cellular engineering.
The developed system offers enhanced control over biological processes in living cells.