Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila

Genetics. 2014 Apr;196(4):961-71. doi: 10.1534/genetics.113.160713. Epub 2014 Jan 29.

Abstract

We and others recently demonstrated that the readily programmable CRISPR/Cas9 system can be used to edit the Drosophila genome. However, most applications to date have relied on aberrant DNA repair to stochastically generate frameshifting indels and adoption has been limited by a lack of tools for efficient identification of targeted events. Here we report optimized tools and techniques for expanded application of the CRISPR/Cas9 system in Drosophila through homology-directed repair (HDR) with double-stranded DNA (dsDNA) donor templates that facilitate complex genome engineering through the precise incorporation of large DNA sequences, including screenable markers. Using these donors, we demonstrate the replacement of a gene with exogenous sequences and the generation of a conditional allele. To optimize efficiency and specificity, we generated transgenic flies that express Cas9 in the germline and directly compared HDR and off-target cleavage rates of different approaches for delivering CRISPR components. We also investigated HDR efficiency in a mutant background previously demonstrated to bias DNA repair toward HDR. Finally, we developed a web-based tool that identifies CRISPR target sites and evaluates their potential for off-target cleavage using empirically rooted rules. Overall, we have found that injection of a dsDNA donor and guide RNA-encoding plasmids into vasa-Cas9 flies yields the highest efficiency HDR and that target sites can be selected to avoid off-target mutations. Efficient and specific CRISPR/Cas9-mediated HDR opens the door to a broad array of complex genome modifications and greatly expands the utility of CRISPR technology for Drosophila research.

Keywords: CRISPR RNA; Cas9; Drosophila; genome engineering; homologous recombination.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • Bacterial Proteins / genetics*
  • CRISPR-Associated Protein 9
  • CRISPR-Associated Proteins / genetics
  • CRISPR-Associated Proteins / metabolism*
  • CRISPR-Cas Systems*
  • DEAD-box RNA Helicases / genetics
  • DNA / metabolism
  • Drosophila Proteins / genetics
  • Drosophila melanogaster / embryology
  • Drosophila melanogaster / genetics*
  • Drosophila melanogaster / metabolism
  • Endonucleases / genetics*
  • Female
  • Gene Editing / methods*
  • Germ Cells / metabolism
  • Male
  • RNA, Guide / metabolism
  • Recombinational DNA Repair*

Substances

  • Bacterial Proteins
  • CRISPR-Associated Proteins
  • Drosophila Proteins
  • RNA, Guide
  • DNA
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Endonucleases
  • vas protein, Drosophila
  • DEAD-box RNA Helicases