In vivo and in vitro knockout system labelled using fluorescent protein via microhomology-mediated end joining

Shota Katayama, Kota Sato, Toru Nakazawa

Research output: Contribution to journalArticle

Abstract

Gene knockout is important for understanding gene function and genetic disorders. The CRISPR/Cas9 system has great potential to achieve this purpose. However, we cannot distinguish visually whether a gene is knocked out and in how many cells it is knocked out among a population of cells. Here, we developed a new system that enables the labelling of knockout cells with fluorescent protein through microhomology-mediated end joining–based knock-in. Using a combination with recombinant adeno-associated virus, we delivered our system into the retina, where the expression of Staphylococcus aureus Cas9 was driven by a retina ganglion cell (RGC)–specific promoter, and knocked out carnitine acetyltransferase (CAT). We evaluated RGCs and revealed that CAT is required for RGC survival. Furthermore, we applied our system to Keap1 and confirmed that Keap1 is not expressed in fluorescently labelled cells. Our system provides a promising framework for cell type–specific genome editing and fluorescent labelling of gene knockout based on knock-in.

Original languageEnglish
Article numbere201900528
JournalLife Science Alliance
Volume3
Issue number1
DOIs
Publication statusPublished - 2020 Jan 1

ASJC Scopus subject areas

  • Ecology
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)
  • Plant Science
  • Health, Toxicology and Mutagenesis

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