Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases

Nat Commun. 2012;3:1017. doi: 10.1038/ncomms2020.

Abstract

Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically relatively basal and comprise many pests. However, the absence of a sophisticated genetic model system, or targeted gene-manipulation system, has limited research on hemimetabolous species. Here we use zinc-finger nuclease and transcription activator-like effector nuclease technologies to produce genetic knockouts in the hemimetabolous insect Gryllus bimaculatus. Following the microinjection of mRNAs encoding zinc-finger nucleases or transcription activator-like effector nucleases into cricket embryos, targeting of a transgene or endogenous gene results in sequence-specific mutations. Up to 48% of founder animals transmit disrupted gene alleles after zinc-finger nucleases microinjection compared with 17% after microinjection of transcription activator-like effector nucleases. Heterozygous offspring is selected using mutation detection assays that use a Surveyor (Cel-I) nuclease, and subsequent sibling crosses create homozygous knockout crickets. This approach is independent from a mutant phenotype or the genetic tractability of the organism of interest and can potentially be applied to manage insect pests using a non-transgenic strategy.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Animals
  • Base Sequence
  • Deoxyribonucleases / chemistry
  • Deoxyribonucleases / genetics
  • Deoxyribonucleases / metabolism*
  • Gene Knockout Techniques / methods*
  • Genome, Insect*
  • Gryllidae / chemistry
  • Gryllidae / enzymology*
  • Gryllidae / genetics*
  • Insect Proteins / chemistry
  • Insect Proteins / genetics
  • Insect Proteins / metabolism*
  • Microinjections
  • Molecular Sequence Data
  • Zinc Fingers

Substances

  • Insect Proteins
  • Deoxyribonucleases