KN
Kohei Nishimura
  • Research scientist, Department of Biological Science, Division of Natural Science, Graduate School of Science, Nagoya University, Japan
A Single-step Generation of AlissAID-based Conditional Knockdown Strains Using Nanobody that Targets GFP or mCherry in Budding Yeast
Authors:  Yoshitaka Ogawa, Taisei P. Ueda, Keisuke Obara, Kohei Nishimura and Takumi Kamura, date: 06/20/2024, view: 660, Q&A: 0

The Auxin-inducible degron (AID) system is a genetic tool that induces rapid target protein depletion in an auxin-dependent manner. Recently, two advanced AID systems—the super-sensitive AID and AID 2—were developed using an improved pair of synthetic auxins and mutated TIR1 proteins. In these AID systems, a nanomolar concentration of synthetic auxins is sufficient as a degradation inducer for target proteins. However, despite these advancements, AID systems still require the fusion of an AID tag to the target protein for degradation, potentially affecting its function and stability. To address this limitation, we developed an affinity linker–based super-sensitive AID (AlissAID) system using a single peptide antibody known as a nanobody. In this system, the degradation of GFP- or mCherry-tagged target proteins is induced in a synthetic auxin (5-Ad-IAA)–dependent manner. Here, we introduce a simple method for generating AlissAID strains targeting GFP or mCherry fusion proteins in budding yeasts.

A Simple Method to Generate Super-sensitive AID (ssAID)-based Conditional Knockouts using CRISPR-based Gene Knockout in Various Vertebrate Cell Lines
Authors:  Kohei Nishimura and Tatsuo Fukagawa, date: 07/20/2021, view: 2735, Q&A: 0

Inducing loss of function of a target protein using methods such as gene knockout is a powerful and useful strategy for analyzing protein function in cells. In recent years, the CRISPR/Cas-9-based gene knockout technology has been widely used across a variety of eukaryotes; however, this type of simple gene knockout strategy is not applicable to essential genes, which require a conditional knockout system. The auxin-inducible degron (AID) system enables rapid depletion of the target protein in an auxin-dependent manner and has been used to generate conditional mutants in various eukaryotic cell lines. One problem with the AID system is the use of high auxin concentrations for protein degradation, which can cause cytotoxicity. Recently, we established a super-sensitive AID (ssAID) system that allowed a reduction in the amount of auxin required by more than 1,000-fold. We also utilized a single-step method to generate AID-based conditional knockout cells with a ssAID system in various cell lines. In this protocol, we introduce our improved method, which provides a powerful tool for the investigation of the roles of essential genes.

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