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Chromosome Research

, Volume 25, Issue 3–4, pp 253–260 | Cite as

An efficient method to generate conditional knockout cell lines for essential genes by combination of auxin-inducible degron tag and CRISPR/Cas9

  • Kohei Nishimura
  • Tatsuo FukagawaEmail author
Original Article

Abstract

Generation of cells with a loss-of-function mutation in a gene (knockout cells) is a valuable technique for studying the function of a given gene product. However, if the product of the target gene is essential for cell viability, conditional knockout cell lines must be generated. Recently, as gene editing technology using CRISPR/Cas9 has developed, it has become possible to produce conditional knockout cell lines using this technique. However, to obtain final conditional knockout cell lines, it is necessary to perform several experiments with multiple complicated steps. In this paper, we introduce an easy and efficient method to generate conditional knockout cell lines based on combining auxin-inducible degron (AID) technology with CRISPR/Cas9 gene editing. Our method only requires performing a single transfection and is therefore an easy and rapid method to obtain a conditional knockout cell line.

Keywords

Aid CRISPR/Cas9 Rapid degradation 

Abbreviations

AID

Auxin-inducible degron

CRISPR

Clustered regularly interspaced short palindromic repeat

Cas9

CRISPR-associated protein 9

sgRNA

Single-guide RNA

CENP

Centromere protein

Notes

Acknowledgments

We are very grateful to R. Fukuoka and Y. Fukagawa for their technical assistance. This work was supported by MEXT KAKENHI Grant Numbers 25221106 and 15H05972 to TF and MEXT KAKENHI Grant Number 17K15041 to KN.

Supplementary material

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Figure S3 (PDF 1051 kb).
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Figure S4 (PDF 2049 kb).

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Copyright information

© Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  1. 1.Graduate School of Frontier BiosciencesOsaka UniversitySuitaJapan

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