A genome editing vector that enables easy selection and identification of knockout cells

Akira Nagasaki, Yoshio Kato, Keiichi Meguro, Ayana Yamagishi, Chikashi Nakamura, Taro Q.P. Uyeda

Research output: Contribution to journalArticlepeer-review

5 Citations (Scopus)

Abstract

The CRISPR/Cas9 system is a powerful genome editing tool for disrupting the expression of specific genes in a variety of cells. However, the genome editing procedure using currently available vectors is laborious, and there is room for improvement to obtain knockout cells more efficiently. Therefore, we constructed a novel vector for high efficiency genome editing, named pGedit, which contains EGFP-Bsr as a selection marker, expression units of Cas9, and sgRNA without a terminator sequence of the U6 promoter. EGFP-Bsr is a fusion protein of EGFP and blasticidin S deaminase, and enables rapid selection and monitoring of transformants, as well as confirmation that the vector has not been integrated into the genome. By using pGedit, we targeted human ACTB, ACTG1 and mouse Nes genes coding for β-actin, γ-actin and nestin, respectively. Knockout cell lines of each gene were easily and efficiently obtained in all three cases. In this report, we show that our novel vector, pGedit, significantly facilitates genome editing.

Original languageEnglish
Pages (from-to)37-44
Number of pages8
JournalPlasmid
Volume98
DOIs
Publication statusPublished - 2018 Jun

Keywords

  • Blasticidin S
  • CRISPR/Cas9
  • GFP
  • Knockout and actin

ASJC Scopus subject areas

  • Molecular Biology

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