Augmenting CRISPR applications in Drosophila with tRNA-flanked sgRNAs

Nat Methods. 2016 Oct;13(10):852-4. doi: 10.1038/nmeth.3972. Epub 2016 Sep 5.

Abstract

We present tRNA-based vectors for producing multiple clustered regularly interspaced short palindromic repeats (CRISPR) single guide RNAs (sgRNAs) from a single RNA polymerase II or III transcript in Drosophila. The system, which is based on liberation of sgRNAs by processing flanking tRNAs, permits highly efficient multiplexing of Cas9-based mutagenesis. We also demonstrate that the tRNA-sgRNA system markedly increases the efficacy of conditional gene disruption by Cas9 and can promote editing by the recently discovered RNA-guided endonuclease Cpf1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Genetically Modified / genetics
  • CRISPR-Associated Proteins / genetics*
  • CRISPR-Cas Systems / genetics
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • Drosophila / enzymology
  • Drosophila / genetics*
  • Mutagenesis, Site-Directed
  • Plasmids
  • RNA Editing / genetics
  • RNA Polymerase II / genetics
  • RNA Polymerase III / genetics
  • RNA, Guide / genetics*
  • RNA, Transfer / genetics*
  • Transcription, Genetic

Substances

  • CRISPR-Associated Proteins
  • RNA, Guide
  • RNA, Transfer
  • RNA Polymerase II
  • RNA Polymerase III