Efficient gene knock-out and knock-in with transgenic Cas9 in Drosophila

G3 (Bethesda). 2014 Mar 21;4(5):925-9. doi: 10.1534/g3.114.010496.

Abstract

Bacterial Cas9 nuclease induces site-specific DNA breaks using small gRNA as guides. Cas9 has been successfully introduced into Drosophila for genome editing. Here, we improve the versatility of this method by developing a transgenic system that expresses Cas9 in the Drosophila germline. Using this system, we induced inheritable knock-out mutations by injecting only the gRNA into embryos, achieved highly efficient mutagenesis by expressing gRNA from the promoter of a novel non-coding RNA gene, and recovered homologous recombination-based knock-in of a fluorescent marker at a rate of 4.5% by co-injecting gRNA with a circular DNA donor.

Keywords: Cas9; Drosophila; gRNA; knock-in; knock-out.

Publication types

  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Genetically Modified*
  • Bacterial Proteins / genetics*
  • Deoxyribonucleases / genetics*
  • Drosophila / genetics*
  • Gene Expression
  • Gene Knock-In Techniques
  • Gene Knockout Techniques
  • Gene Order
  • Gene Targeting
  • Germ-Line Mutation
  • Homologous Recombination
  • Microinjections
  • RNA, Guide, CRISPR-Cas Systems
  • Transgenes

Substances

  • Bacterial Proteins
  • Deoxyribonucleases